Methods for treatment of polycystic kidney disease

ABSTRACT

Provided herein are methods for the treatment of polycystic kidney disease, including autosomal dominant polycystic kidney disease, using modified oligonucleotides targeted to miR-17.

This application is a continuation of U.S. application Ser. No.16/823,904, filed Mar. 19, 2020, which is a continuation of U.S.application Ser. No. 15/753,865, filed Feb. 20, 2018, now U.S. Pat. No.10,633,657, which is a national stage application of InternationalApplication No. PCT/US2016/048603, filed Aug. 25, 2016, which claims thebenefit of priority of U.S. Provisional Application No. 62/210,031,filed Aug. 26, 2015, each of which is incorporated by reference hereinin its entirety for any purpose.

The sequence listing that is contained in the file named“RGLSP0002USC2.txt”, which is 8 KB (as measured in Microsoft Windows®)and was created on Oct. 4, 2021, is filed herewith by electronicsubmission and is incorporated by reference herein.

FIELD OF INVENTION

Provided herein are methods and compositions for the treatment ofpolycystic kidney disease.

BACKGROUND

Polycystic kidney disease is a genetic disorder in which multiplefluid-filled cysts develop in the kidneys, and elsewhere in the body.Polycystic kidney disease can be inherited as autosomal recessive(ARPKD) or autosomal dominant (ADPKD). Autosomal dominant polycystickidney disease is caused by mutations in the PKD1 or PKD2 gene. ADPKD isa progressive disease in which cyst formation and renal enlargement leadto renal insufficiency and eventually end-stage renal disease in 50% ofpatients by age 60. ADPKD patients may require lifelong dialysis and/orkidney transplant. There is currently no approved therapeutic agent fortreating ADPKD.

SUMMARY OF INVENTION

Provided here are methods for treating polycystic kidney diseasecomprising administering to a subject in need thereof a therapeuticallyeffective amount of a compound comprising a modified oligonucleotideconsisting of 8 to 25 linked nucleosides, wherein the nucleobasesequence of the modified oligonucleotide is complementary to miR-17. Incertain embodiments, the subject has polycystic kidney disease. Incertain embodiments, the subject is suspected of having polycystickidney disease.

In certain embodiments, the subject has been diagnosed as havingpolycystic kidney disease prior to administering the compound comprisingthe modified oligonucleotide. In some embodiments, the subject, prior toadministration of the compound comprising the modified oligonucleotide,was determined to have an increased level of miR-17 in the kidney, urineor blood of the subject.

In certain embodiments, the polycystic kidney disease is autosomaldominant polycystic kidney disease (ADPKD). In some embodiments, thepolycystic kidney disease is autosomal recessive polycystic kidneydisease (ARPKD). In some embodiments, the subject has a mutation in thePKD1 gene. In some embodiments, the subject has a mutation in the PKD2gene.

In certain embodiments, the subject has increased total kidney volume.In some embodiments, the subject has hypertension. In some embodiments,the subject has impaired kidney function. In some embodiments, thesubject is in need of improved kidney function.

In any of the embodiments provided herein, administration of a compoundcomprising a modified oligonucleotide complementary to miR-17, to asubject having polycystic kidney disease, may improve kidney function inthe subject; delay the worsening of kidney function in the subject;reduce total kidney volume in the subject; slow the increase in totalkidney volume in the subject; inhibit cyst growth in the subject; slowthe increase in cyst growth in the subject; reduce kidney pain in thesubject; slow the increase in kidney pain in the subject; delay theonset of kidney pain in the subject; reduce hypertension in the subject;slow the worsening of hypertension in the subject; delay the onset ofhypertension in the subject; reduce fibrosis in the kidney of thesubject; slow the worsening of fibrosis in the kidney of the subject;delay the onset of end stage renal disease in the subject; delay time todialysis for the subject; delay time to renal transplant for thesubject; and/or improve life expectancy of the subject.

In any of the embodiments provided herein, administration of a compoundcomprising a modified oligonucleotide complementary to miR-17, to asubject having polycystic kidney disease, may reduce albuminuria in thesubject; slow the worsening of albuminuria in the subject; delay theonset of albuminuria in the subject; reduce hematuria in the subject;slow the worsening of hematuria in the subject; delay the onset ofhematuria in the subject; reduces blood urea nitrogen in the subject;reduce creatinine in the blood of the subject; improve creatinineclearance in the subject; reduce albumin:creatinine ratio in thesubject; improve glomerular filtration rate in the subject; slows theworsening of glomerular filtration rate in the subject; reduceneutrophil gelatinase-associated lipocalin (NGAL) protein in the urineof the subject; and/or reduce kidney injury molecule-1 (KIM-1) proteinin the urine of the subject.

Any of the embodiments provided herein may comprise measuring totalkidney volume in the subject; measuring hypertension in the subject;measuring kidney pain in the subject; measuring fibrosis in the kidneyof the subject; measuring blood urea nitrogen in the blood of thesubject; measuring creatinine in the blood of the subject; measuringcreatinine clearance in the subject; measuring albuminuria in thesubject; measuring albumin:creatinine ratio in the subject; measuringglomerular filtration rate in the subject; measuring neutrophilgelatinase-associated lipocalin (NGAL) protein in the urine of thesubject; and/or measuring kidney injury molecule-1 (KIM-1) protein inthe urine of the subject.

In certain embodiments, the total kidney volume is height-adjustedkidney volume.

In certain embodiments, a cyst is present in the kidney of a subject. Insome embodiments, a cyst is present in the kidney and liver of asubject.

Any of the embodiments provided herein may comprise administering atleast one additional therapy that is an anti-hypertensive agent.

Any of the embodiments provided herein may comprise administering atleast one additional therapy selected from an angiotensin II convertingenzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), adiuretic, a calcium channel blocker, a kinase inhibitor, an adrenergicreceptor antagonist, a vasodilator, a benzodiazepine, a renin inhibitor,an aldosterone receptor antagonist, an endothelin receptor blocker, anmammalian target of rapamycin (mTOR) inhibitor, a hormone analogue, avasopressin receptor 2 antagonist, an aldosterone receptor antagonist,dialysis, and kidney transplant.

In certain embodiments, a vasopressin receptor 2 antagonist istolvaptan.

In certain embodiments, the angiotensin II converting enzyme (ACE)inhibitors is selected from captopril, enalapril, lisinopril,benazepril, quinapril, fosinopril, ramipril, cilazapril, perindopril,and trandolapril.

In certain embodiments, the angiotensin II receptor blockers (ARB) isselected from candesartan, irbesartan, olmesartan, losartan, valsartan,telmisartan, and eprosartan.

In certain embodiments, an ACE inhibitor is administered at a doseranging from 0.5 to 1 mg/m²/day, from 1 to 6 mg/m²/day, from 1 to 2mg/m²/day, from 2 to 4 mg/m²/day, or from 4 to 8 mg/m²/day.

In certain embodiments, an ARB is administered at a dose ranging from6.25 to 150 mg/m2/day. In any of these embodiments, an ARB isadministered at a dose of 6.25 mg/m²/day, 10 mg/m²/day, 12.5 mg/m²/day,18.75 mg/m²/day, 37.5 mg/m²/day, 50 mg/m²/day, or 150 mg/m²/day.

In certain embodiments, the at least one additional therapy is analdosterone receptor antagonist. In certain embodiments, an aldosteronereceptor antagonist is spironolactone. In certain embodiments,spironolactone is administered at a dose ranging from 10 to 35 mg daily.In certain embodiments, spironolactone is administered at a dose of 25mg daily.

In certain embodiments, a kinase inhibitor is selected from bosutiniband KD019.

In certain embodiments, an mTOR inhibitor is selected from everolimus,rapamycin, and sirolimus.

In certain embodiments, a hormone analogue is selected from somatostatinand adrenocorticotrophic hormone.

In any of the embodiments provided herein, the nucleobase sequence ofthe modified oligonucleotide is at least 90% complementary, is at least95% complementary, or is 100% complementary to the nucleobase sequenceof miR-17 (SEQ ID NO: 1).

In any of the embodiments provided herein, the nucleobase sequence ofthe modified oligonucleotide comprises the nucleobase sequence5′-GCACTTTG-3′ (SEQ ID NO: 3), wherein each T in the nucleobase sequenceis independently selected from a T and a U.

In any of the embodiments provided herein, the modified oligonucleotideconsists of 8 to 25, 8 to 12, 12 to 25, 15 to 25, or 17 to 23 linkednucleosides. In any of the embodiments provided herein, the modifiedoligonucleotide consists of 8, 9, 10, 11 or 12 linked nucleosides. Inany of the embodiments provided herein, the modified oligonucleotideconsists of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25linked nucleosides. In any of the embodiments provided herein, themodified oligonucleotide consists of 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25 linked nucleosides. In any of the embodiments provided herein,the modified oligonucleotide consists of 17, 18, 19, 20, 21, 22, or 23linked nucleosides.

In any of the embodiments provided herein, the modified oligonucleotidecomprises at least one modified nucleoside. The modified nucleoside maybe selected from an S-cEt nucleoside, a 2′-O-methoxyethyl nucleoside,and an LNA nucleoside. The modified oligonucleotide may comprise atleast one modified internucleoside linkage. Each internucleoside linkageof the modified oligonucleotide may be a modified internucleosidelinkage. In certain embodiments, the modified internucleoside linkage isa phosphorothioate internucleoside linkage.

In any of the embodiments provided herein, the compound consists of themodified oligonucleotide.

In any of the embodiments provided herein, a therapeutically effectiveamount of the compound comprising a modified oligonucleotidecomplementary to miR-17 is administered to the subject.

Provided herein is the use of a compound comprising a modifiedoligonucleotide consisting of 8 to 25 linked nucleosides, wherein thenucleobase sequence of the modified oligonucleotide is complementary tomiR-17, for the treatment of polycystic kidney disease.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A-B. (A) Genomic organization of the miR-17-92 and its paralogousclusters miR-106a˜363 and miR-106b˜25; (B) miR-17, miR-18, miR-19, andmiR-92 microRNA families.

FIG. 2A-B. Treatment of Pcy mice with anti-miR-17 leads to (A) reductionin kidney weight to body weight ratio and (B) cystic index.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in thearts to which the invention belongs. Unless specific definitions areprovided, the nomenclature utilized in connection with, and theprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well-known and commonly used in the art. In the event thatthere is a plurality of definitions for terms herein, those in thissection prevail. Standard techniques may be used for chemical synthesis,chemical analysis, pharmaceutical preparation, formulation and delivery,and treatment of subjects. Certain such techniques and procedures may befound for example in “Carbohydrate Modifications in Antisense Research”Edited by Sangvi and Cook, American Chemical Society, Washington D.C.,1994; and “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., 18th edition, 1990; and which is hereby incorporated byreference for any purpose. Where permitted, all patents, patentapplications, published applications and publications, GENBANKsequences, websites and other published materials referred to throughoutthe entire disclosure herein, unless noted otherwise, are incorporatedby reference in their entirety. Where reference is made to a URL orother such identifier or address, it is understood that such identifierscan change and particular information on the internet can change, butequivalent information can be found by searching the internet. Referencethereto evidences the availability and public dissemination of suchinformation.

Before the present compositions and methods are disclosed and described,it is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

Definitions

“Polycystic kidney disease” or “PKD” is an inherited form of kidneydisease in which multiple cysts form in at least one kidney, leading toenlargement of the affected kidney(s) and progressive loss of kidneyfunction.

“Autosomal dominant polycystic kidney disease” or “ADPKD” is typicallycaused by one or more genetic mutations in the PKD1 and/or PKD2 gene.85% of ADPKD is caused by mutations in PKD1, which is located onchromosome 16, with the majority of the remaining ADPKD cases caused bymutations in PKD2, which is located on chromosome 4.

“Autosomal recessive polycystic kidney disease” or “ARPKD” is typicallycaused by one or more genetic mutations in the PKHD1 gene, which islocated on chromosome 6. Up to 50% of neonates with ARPKD die fromcomplications of intrauterine kidney disease, and about a third of thosewho survive develop end stage renal disease (ESRD) within 10 years.

“Total kidney volume” or “TKV” is a measurement of total kidney volumewhich may be determined by Magnetic Resonance Imaging (MRI), ComputedTomography (CT) scan, or ultrasound (US) imaging, and the volumecalculated by a standard methodology, such as an ellipsoid volumeequation (for ultrasound), or by quantitative stereology or boundarytracing (for CT/MRI). TKV generally increases steadily in ADPKDpatients, with increases correlating with a decline in kidney function.

“Height-adjusted total kidney volume” or “HtTKV” is a measure of totalkidney volume per unit height. Patients with an HtTKV value ≥600 ml/mare predicted to develop stage 3 chronic kidney disease within 8 years.

“Kidney pain” means clinically significant kidney pain necessitatingmedical leave, pharmacologic treatment (narcotic or last-resortanalgesic agents), or invasive intervention.

“Worsening hypertension” means a change in blood pressure that requiresan increase in hypertensive treatment.

“Fibrosis” means the formation or development of excess fibrousconnective tissue in an organ or tissue. In certain embodiments,fibrosis occurs as a reparative or reactive process. In certainembodiments, fibrosis occurs in response to damage or injury. The term“fibrosis” is to be understood as the formation or development of excessfibrous connective tissue in an organ or tissue as a reparative orreactive process, as opposed to a formation of fibrous tissue as anormal constituent of an organ or tissue

“Hematuria” means the presence of red blood cells in the urine.

“Albuminuria” means the presence of excess albumin in the urine, andincludes without limitation, normal albuminuria, high normalalbuminuria, microalbuminuria and macroalbuminuria. Normally, theglomerular filtration permeability barrier, which is composed ofpodocyte, glomerular basement membrane and endothelial cells, preventsserum protein from leaking into urine. Albuminuria may reflect injury ofthe glomerular filtration permeability barrier. Albuminuria may becalculated from a 24-hour urine sample, an overnight urine sample or aspot-urine sample.

“High normal albuminuria” means elevated albuminuria characterized by(i) the excretion of 15 to <30 mg of albumin into the urine per 24 hoursand/or (ii) an albumin/creatinine ratio of 1.25 to <2.5 mg/mmol (or 10to <20 mg/g) in males or 1.75 to <3.5 mg/mmol (or 15 to <30 mg/g) infemales.

“Microalbuminuria” means elevated albuminuria characterized by (i) theexcretion of 30 to 300 mg of albumin into the urine per 24 hours and/or(ii) an albumin/creatinine ratio of 2.5 to <25 mg/mmol (or 20 to <200mg/g) in males or 3.5 to <35 mg/mmol (or 30 to <300 mg/g) in females.

“Macroalbuminuria” means elevated albuminuria characterized by theexcretion of more than 300 mg of albumin into the urine per 24 hoursand/or (ii) an albumin/creatinine ratio of >25 mg/mmol (or >200 mg/g) inmales or >35 mg/mmol (or >300 mg/g) in females.

“Albumin/creatinine ratio” means the ratio of urine albumin (mg/dL) perurine creatinine (g/dL) and is expressed as mg/g. In certainembodiments, albumin/creatinine ratio may be calculated from aspot-urine sample and may be used as an estimate of albumin excretionover a 24 hour period.

“Estimated glomerular filtration rate (eGFR) or “glomerular filtrationrate (GFR)” means a measurement of how well the kidneys are filteringcreatinine, and is used as an estimate of how much blood passes throughthe glomeruli per minute. Normal results may range from 90-120mL/min/1.73 m². Levels below 60 mL/min/1.73 m² for 3 or more months maybe an indicator chronic kidney disease. Levels below 15 mL/min/1.73 m²may be an indicator of kidney failure.

“Proteinuria” means the presence of an excess of serum proteins in theurine. Proteinuria may be characterized by the excretion of >250 mg ofprotein into the urine per 24 hours and/or a urine protein to creatinineratio of >0.20 mg/mg. Serum proteins elevated in association withproteinuria include, without limitation, albumin.

“Blood urea nitrogen” or “BUN” means a measure of the amount of nitrogenin the blood in the form of urea. The liver produces urea in the ureacycle as a waste product of the digestion of protein, and the urea isremoved from the blood by the kidneys. Normal human adult blood maycontain between 7 to 21 mg of urea nitrogen per 100 ml (7-21 mg/dL) ofblood. Measurement of blood urea nitrogen is used as an indicator ofrenal health. If the kidneys are not able to remove urea from the bloodnormally, a subject's BUN rises.

“End stage renal disease (ESRD)” means the complete or almost completefailure of kidney function.

“Impaired kidney function” means reduced kidney function, relative tonormal kidney function.

“Slow the worsening of” and “slow worsening” mean to reduce the rate atwhich a medical condition moves towards an advanced state.

“Delay time to dialysis” means to maintain sufficient kidney functionsuch that the need for dialysis treatment is delayed.

“Delay time to renal transplant” means to maintain sufficient kidneyfunction such that the need for a kidney transplant is delayed.

“Improves life expectancy” means to lengthen the life of a subject bytreating one or more symptoms of a disease in the subject.

“Subject” means a human or non-human animal selected for treatment ortherapy.

“Subject in need thereof” means a subject that is identified as in needof a therapy or treatment.

“Subject suspected of having” means a subject exhibiting one or moreclinical indicators of a disease.

“Administering” means providing a pharmaceutical agent or composition toa subject, and includes, but is not limited to, administering by amedical professional and self-administering.

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes, but is not limited to,subcutaneous administration, intravenous administration, andintramuscular administration.

“Subcutaneous administration” means administration just below the skin.

“Intravenous administration” means administration into a vein.

“Administered concomitantly” refers to the co-administration of two ormore agents in any manner in which the pharmacological effects of bothare manifest in the patient at the same time. Concomitant administrationdoes not require that both agents be administered in a singlepharmaceutical composition, in the same dosage form, or by the sameroute of administration. The effects of both agents need not manifestthemselves at the same time. The effects need only be overlapping for aperiod of time and need not be coextensive.

“Duration” means the period of time during which an activity or eventcontinues. In certain embodiments, the duration of treatment is theperiod of time during which doses of a pharmaceutical agent orpharmaceutical composition are administered.

“Therapy” means a disease treatment method. In certain embodiments,therapy includes, but is not limited to, administration of one or morepharmaceutical agents to a subject having a disease.

“Treat” means to apply one or more specific procedures used for the cureof a disease or the amelioration at least one indicator of a disease. Incertain embodiments, the specific procedure is the administration of oneor more pharmaceutical agents. In certain embodiments, treatment of PKDincludes, but is not limited to, reducing total kidney volume, improvingkidney function, reducing hypertension, and/or reducing kidney pain.

“Ameliorate” means to lessen the severity of at least one indicator of acondition or disease. In certain embodiments, amelioration includes adelay or slowing in the progression of one or more indicators of acondition or disease. The severity of indicators may be determined bysubjective or objective measures which are known to those skilled in theart.

“At risk for developing” means the state in which a subject ispredisposed to developing a condition or disease. In certainembodiments, a subject at risk for developing a condition or diseaseexhibits one or more symptoms of the condition or disease, but does notexhibit a sufficient number of symptoms to be diagnosed with thecondition or disease. In certain embodiments, a subject at risk fordeveloping a condition or disease exhibits one or more symptoms of thecondition or disease, but to a lesser extent required to be diagnosedwith the condition or disease.

“Prevent the onset of” means to prevent the development of a conditionor disease in a subject who is at risk for developing the disease orcondition. In certain embodiments, a subject at risk for developing thedisease or condition receives treatment similar to the treatmentreceived by a subject who already has the disease or condition.

“Delay the onset of” means to delay the development of a condition ordisease in a subject who is at risk for developing the disease orcondition. In certain embodiments, a subject at risk for developing thedisease or condition receives treatment similar to the treatmentreceived by a subject who already has the disease or condition.

“Therapeutic agent” means a pharmaceutical agent used for the cure,amelioration or prevention of a disease.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration. In certain embodiments, a dose may beadministered in two or more boluses, tablets, or injections. Forexample, in certain embodiments, where subcutaneous administration isdesired, the desired dose requires a volume not easily accommodated by asingle injection. In such embodiments, two or more injections may beused to achieve the desired dose. In certain embodiments, a dose may beadministered in two or more injections to minimize injection sitereaction in an individual. In certain embodiments, a dose isadministered as a slow infusion.

“Dosage unit” means a form in which a pharmaceutical agent is provided.In certain embodiments, a dosage unit is a vial containing lyophilizedoligonucleotide. In certain embodiments, a dosage unit is a vialcontaining reconstituted oligonucleotide.

“Therapeutically effective amount” refers to an amount of apharmaceutical agent that provides a therapeutic benefit to an animal.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual that includes a pharmaceutical agent. Forexample, a pharmaceutical composition may comprise a sterile aqueoussolution.

“Pharmaceutical agent” means a substance that provides a therapeuticeffect when administered to a subject.

“Active pharmaceutical ingredient” means the substance in apharmaceutical composition that provides a desired effect.

“Pharmaceutically acceptable salt” means a physiologically andpharmaceutically acceptable salt of a compound provided herein, i.e., asalt that retains the desired biological activity of the compound anddoes not have undesired toxicological effects when administered to asubject. Nonlimiting exemplary pharmaceutically acceptable salts ofcompounds provided herein include sodium and potassium salt forms. Theterm “compound” as used herein includes pharmaceutically acceptablesalts thereof unless specifically indicated otherwise.

“Improved organ function” means a change in organ function toward normallimits. In certain embodiments, organ function is assessed by measuringmolecules found in a subject's blood or urine. For example, in certainembodiments, improved kidney function is measured by a reduction inblood urea nitrogen, a reduction in proteinuria, a reduction inalbuminuria, etc.

“Acceptable safety profile” means a pattern of side effects that iswithin clinically acceptable limits.

“Side effect” means a physiological response attributable to a treatmentother than desired effects. In certain embodiments, side effectsinclude, without limitation, injection site reactions, liver functiontest abnormalities, kidney function abnormalities, liver toxicity, renaltoxicity, central nervous system abnormalities, and myopathies. Suchside effects may be detected directly or indirectly. For example,increased aminotransferase levels in serum may indicate liver toxicityor liver function abnormality. For example, increased bilirubin mayindicate liver toxicity or liver function abnormality.

“Subject compliance” means adherence to a recommended or prescribedtherapy by a subject.

“Comply” means the adherence with a recommended therapy by a subject.

“Recommended therapy” means a treatment recommended by a medicalprofessional for the treatment, amelioration, or prevention of adisease.

The term “blood” as used herein, encompasses whole blood and bloodfractions, such as serum and plasma.

“Anti-miR” means an oligonucleotide having a nucleobase sequencecomplementary to a microRNA. In certain embodiments, an anti-miR is amodified oligonucleotide.

“Anti-miR-X” where “miR-X” designates a particular microRNA, means anoligonucleotide having a nucleobase sequence complementary to miR-X. Incertain embodiments, an anti-miR-X is fully complementary (i.e., 100%complementary) to miR-X. In certain embodiments, an anti-miR-X is atleast 80%, at least 85%, at least 90%, or at least 95% complementary tomiR-X. In certain embodiments, an anti-miR-X is a modifiedoligonucleotide.

“miR-17” means the mature miRNA having the nucleobase sequence

(SEQ ID NO: 1) CAAAGUGCUUACAGUGCAGGUAG.

“miR-17 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence

(SEQ ID NO: 2) GUCAGAAUAAUGUCAAAGUGCUUACAGUGCAGGUAGUGAUAUGUGCAUCUACUGCAGUGAAGGCACUUGUAGCAUUAUGGUGAC.

“miR-17 2-7 seed sequence” means the nucleobase sequence from positions2 to 7 of

SEQ ID NO: 1 AAAGUG.

“miR-17 family member” means a mature miRNA having a nucleobase sequencecomprising the miR-17 2-7 seed sequence.

“miR-17 family” means a group of miRNAs, each having a nucleobasesequence comprising the miR-17 2-7 seed sequence.

“Target nucleic acid” means a nucleic acid to which an oligomericcompound is designed to hybridize.

“Targeting” means the process of design and selection of nucleobasesequence that will hybridize to a target nucleic acid.

“Targeted to” means having a nucleobase sequence that will allowhybridization to a target nucleic acid.

“Modulation” means a perturbation of function, amount, or activity. Incertain embodiments, modulation means an increase in function, amount,or activity. In certain embodiments, modulation means a decrease infunction, amount, or activity.

“Expression” means any functions and steps by which a gene's codedinformation is converted into structures present and operating in acell.

“Nucleobase sequence” means the order of contiguous nucleobases in anoligomeric compound or nucleic acid, typically listed in a 5′ to 3′orientation, independent of any sugar, linkage, and/or nucleobasemodification.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother in a nucleic acid.

“Nucleobase complementarity” means the ability of two nucleobases topair non-covalently via hydrogen bonding.

“Complementary” means that one nucleic acid is capable of hybridizing toanother nucleic acid or oligonucleotide. In certain embodiments,complementary refers to an oligonucleotide capable of hybridizing to atarget nucleic acid.

“Fully complementary” means each nucleobase of an oligonucleotide iscapable of pairing with a nucleobase at each corresponding position in atarget nucleic acid. In certain embodiments, an oligonucleotide is fullycomplementary to a microRNA, i.e. each nucleobase of the oligonucleotideis complementary to a nucleobase at a corresponding position in themicroRNA. A modified oligonucleotide may be fully complementary to amicroRNA, and have a number of linked nucleosides that is less than thelength of the microRNA. For example, an oligonucleotide with 16 linkednucleosides, where each nucleobase of the oligonucleotide iscomplementary to a nucleobase at a corresponding position in a microRNA,is fully complementary to the microRNA. In certain embodiments, anoligonucleotide wherein each nucleobase has complementarity to anucleobase within a region of a microRNA stem-loop sequence is fullycomplementary to the microRNA stem-loop sequence.

“Percent complementarity” means the percentage of nucleobases of anoligonucleotide that are complementary to an equal-length portion of atarget nucleic acid. Percent complementarity is calculated by dividingthe number of nucleobases of the oligonucleotide that are complementaryto nucleobases at corresponding positions in the target nucleic acid bythe total number of nucleobases in the oligonucleotide.

“Percent identity” means the number of nucleobases in a first nucleicacid that are identical to nucleobases at corresponding positions in asecond nucleic acid, divided by the total number of nucleobases in thefirst nucleic acid. In certain embodiments, the first nucleic acid is amicroRNA and the second nucleic acid is a microRNA. In certainembodiments, the first nucleic acid is an oligonucleotide and the secondnucleic acid is an oligonucleotide.

“Hybridize” means the annealing of complementary nucleic acids thatoccurs through nucleobase complementarity.

“Mismatch” means a nucleobase of a first nucleic acid that is notcapable of Watson-Crick pairing with a nucleobase at a correspondingposition of a second nucleic acid.

“Identical” in the context of nucleobase sequences, means having thesame nucleobase sequence, independent of sugar, linkage, and/ornucleobase modifications and independent of the methyl state of anypyrimidines present.

“MicroRNA” means an endogenous non-coding RNA between 18 and 25nucleobases in length, which is the product of cleavage of apre-microRNA by the enzyme Dicer. Examples of mature microRNAs are foundin the microRNA database known as miRBase(http://microrna.sanger.ac.uk/). In certain embodiments, microRNA isabbreviated as “microRNA” or “miR.”

“Pre-microRNA” or “pre-miR” means a non-coding RNA having a hairpinstructure, which is the product of cleavage of a pri-miR by thedouble-stranded RNA-specific ribonuclease known as Drosha.

“Stem-loop sequence” means an RNA having a hairpin structure andcontaining a mature microRNA sequence. Pre-microRNA sequences andstem-loop sequences may overlap. Examples of stem-loop sequences arefound in the microRNA database known as miRBase(http://microrna.sanger.ac.uk/).

“Pri-microRNA” or “pri-miR” means a non-coding RNA having a hairpinstructure that is a substrate for the double-stranded RNA-specificribonuclease Drosha.

“microRNA precursor” means a transcript that originates from a genomicDNA and that comprises a non-coding, structured RNA comprising one ormore microRNA sequences. For example, in certain embodiments a microRNAprecursor is a pre-microRNA. In certain embodiments, a microRNAprecursor is a pri-microRNA.

“microRNA-regulated transcript” means a transcript that is regulated bya microRNA.

“Seed sequence” means a nucleobase sequence comprising from 6 to 8contiguous nucleobases of nucleobases 1 to 9 of the 5′-end of a maturemicroRNA sequence.

“Seed match sequence” means a nucleobase sequence that is complementaryto a seed sequence, and is the same length as the seed sequence.

“Oligomeric compound” means a compound that comprises a plurality oflinked monomeric subunits. Oligomeric compounds includeoligonucleotides.

“Oligonucleotide” means a compound comprising a plurality of linkednucleosides, each of which can be modified or unmodified, independentfrom one another.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage between nucleosides.

“Natural sugar” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Internucleoside linkage” means a covalent linkage between adjacentnucleosides.

“Linked nucleosides” means nucleosides joined by a covalent linkage.

“Nucleobase” means a heterocyclic moiety capable of non-covalentlypairing with another nucleobase.

“Nucleoside” means a nucleobase linked to a sugar moiety.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of a nucleoside.

“Compound comprising a modified oligonucleotide consisting of” a numberof linked nucleosides means a compound that includes a modifiedoligonucleotide having the specified number of linked nucleosides. Thus,the compound may include additional substituents or conjugates. Unlessotherwise indicated, the compound does not include any additionalnucleosides beyond those of the modified oligonucleotide. For example,unless otherwise indicated, a compound comprising a modifiedoligonucleotide does not include a complementary strand hybridized tothe modified oligonucleotide (i.e., the modified oligonucleotide is asingle-stranded modified oligonucleotide).

“Modified oligonucleotide” means an oligonucleotide having one or moremodifications relative to a naturally occurring terminus, sugar,nucleobase, and/or internucleoside linkage. A modified oligonucleotidemay comprise unmodified nucleosides.

“Single-stranded modified oligonucleotide” means a modifiedoligonucleotide which is not hybridized to a complementary strand.

“Modified nucleoside” means a nucleoside having any change from anaturally occurring nucleoside. A modified nucleoside may have amodified sugar and an unmodified nucleobase. A modified nucleoside mayhave a modified sugar and a modified nucleobase. A modified nucleosidemay have a natural sugar and a modified nucleobase. In certainembodiments, a modified nucleoside is a bicyclic nucleoside. In certainembodiments, a modified nucleoside is a non-bicyclic nucleoside.

“Modified internucleoside linkage” means any change from a naturallyoccurring internucleoside linkage.

“Phosphorothioate internucleoside linkage” means a linkage betweennucleosides where one of the non-bridging atoms is a sulfur atom.

“Modified sugar moiety” means substitution and/or any change from anatural sugar.

“Unmodified nucleobase” means the naturally occurring heterocyclic basesof RNA or DNA: the purine bases adenine (A) and guanine (G), and thepyrimidine bases thymine (T), cytosine (C) (including 5-methylcytosine),and uracil (U).

“5-methylcytosine” means a cytosine comprising a methyl group attachedto the 5 position.

“Non-methylated cytosine” means a cytosine that does not have a methylgroup attached to the 5 position.

“Modified nucleobase” means any nucleobase that is not an unmodifiednucleobase.

“Sugar moiety” means a naturally occurring furanosyl or a modified sugarmoiety.

“Modified sugar moiety” means a substituted sugar moiety or a sugarsurrogate.

“2′-O-methyl sugar” or “2′-OMe sugar” means a sugar having an O-methylmodification at the 2′ position.

“2′-O-methoxyethyl sugar” or “2′-MOE sugar” means a sugar having anO-methoxyethyl modification at the 2′ position.

“2′-fluoro” or “2′-F” means a sugar having a fluoro modification of the2′ position.

“Bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to7 membered ring (including by not limited to a furanosyl) comprising abridge connecting two atoms of the 4 to 7 membered ring to form a secondring, resulting in a bicyclic structure. In certain embodiments, the 4to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7membered ring is a furanosyl. In certain such embodiments, the bridgeconnects the 2′-carbon and the 4′-carbon of the furanosyl. Nonlimitingexemplary bicyclic sugar moieties include LNA, ENA, cEt, S-cEt, andR-cEt.

“Locked nucleic acid (LNA) sugar moiety” means a substituted sugarmoiety comprising a (CH₂)—O bridge between the 4′ and 2′ furanose ringatoms.

“ENA sugar moiety” means a substituted sugar moiety comprising a(CH₂)₂—O bridge between the 4′ and 2′ furanose ring atoms.

“Constrained ethyl (cEt) sugar moiety” means a substituted sugar moietycomprising a CH(CH₃)—O bridge between the 4′ and the 2′ furanose ringatoms. In certain embodiments, the CH(CH₃)—O bridge is constrained inthe S orientation. In certain embodiments, the (CH₂)₂—O is constrainedin the R orientation.

“S-cEt sugar moiety” means a substituted sugar moiety comprising anS-constrained CH(CH₃)—O bridge between the 4′ and the 2′ furanose ringatoms.

“R-cEt sugar moiety” means a substituted sugar moiety comprising anR-constrained CH(CH₃)—O bridge between the 4′ and the 2′ furanose ringatoms.

“2′-O-methyl nucleoside” means a 2′-modified nucleoside having a2′-O-methyl sugar modification.

“2′-O-methoxyethyl nucleoside” means a 2′-modified nucleoside having a2′-O-methoxyethyl sugar modification. A 2′-O-methoxyethyl nucleoside maycomprise a modified or unmodified nucleobase.

“2′-fluoro nucleoside” means a 2′-modified nucleoside having a 2′-fluorosugar modification. A 2′-fluoro nucleoside may comprise a modified orunmodified nucleobase.

“Bicyclic nucleoside” means a 2′-modified nucleoside having a bicyclicsugar moiety. A bicyclic nucleoside may have a modified or unmodifiednucleobase.

“cEt nucleoside” means a nucleoside comprising a cEt sugar moiety. A cEtnucleoside may comprise a modified or unmodified nucleobase.

“S-cEt nucleoside” means a nucleoside comprising an S-cEt sugar moiety.

“R-cEt nucleoside” means a nucleoside comprising an R-cEt sugar moiety.

“β-D-deoxyribonucleoside” means a naturally occurring DNA nucleoside.

“β-D-ribonucleoside” means a naturally occurring RNA nucleoside.

“LNA nucleoside” means a nucleoside comprising a LNA sugar moiety.

“ENA nucleoside” means a nucleoside comprising an ENA sugar moiety.

Overview

Polycystic kidney disease (PKD) is an inherited form of kidney diseasein which fluid-filled cysts develop in the kidneys, leading to kidneyenlargement, renal insufficiency, and often end-stage renal disease. Theexcessive proliferation of cysts is a hallmark pathological feature ofPKD. In the management of PKD, the primary goal for treatment is tomaintain kidney function and prevent the onset of end-stage renaldisease (ESRD), which in turn improves life expectancy of subjects withPKD.

Multiple members of the miR-17-92 cluster of microRNAs are upregulatedin mouse models of PKD. Genetic deletion of the miR-17-92 cluster in amouse model of PKD reduces kidney cyst growth, improves renal function,and prolongs survival (Patel et al., PNAS, 2013; 110(26): 10765-10770).The miR-17-92 cluster contains 6 different microRNAs, each with distinctsequences: miR-17, miR-18a, miR-19a, miR-19-b-1 and miR-92a-1. Thus,genetic deletion of the entire cluster deletes six different microRNAgenes. What this genetic deletion experiment does not demonstrate iswhether inhibition of a subset of microRNAs in the cluster would producethe same improvements in clinical markers of PKD.

It is demonstrated herein that a modified oligonucleotide targeted tomiR-17 improves kidney function and reduces kidney weight in anexperimental model of PKD. Further, miR-17 inhibition also suppressedproliferation and cyst growth of primary cultures derived from cysts ofhuman donors. These data demonstrate that modified oligonucleotidestargeted to miR-17 are useful for the treatment of PKD.

Certain Uses of the Invention

Provided herein are methods for the treatment of polycystic kidneydisease (PKD), comprising administering to a subject having or suspectedof having PKD a compound comprising a modified oligonucleotidecomplementary to miR-17.

In certain embodiments, the subject has been diagnosed as having PKDprior to administration of the compound comprising the modifiedoligonucleotide. Diagnosis of PKD may be achieved through evaluation ofparameters including, without limitation, a subject's family history,clinical features (including without limitation hypertension,albuminuria, hematuria, and impaired GFR), and kidney imaging studies(including without limitation MRI, ultrasound, and CT scan). Diagnosisof PKD may also include screening for mutations in one or more of thePKD1, PKD2, or PKHD1 genes.

The subject having or suspected of having ADPKD may have a mutation inthe PKD1 gene or a mutation in the PKD2 gene. The subject having orsuspected of having ARPKD may have a mutation in the PKHD1 gene.

In certain embodiments the subject has an increased total kidney volume.In certain embodiments, the total kidney volume is height-adjusted totalkidney volume (HtTKV). In certain embodiments, the subject hashypertension. In certain embodiments, the subject has impaired kidneyfunction. In certain embodiments, the subject is in need of improvedkidney function. In certain embodiments, the subject is identified ashaving impaired kidney function.

In certain embodiments, levels of miR-17 are increased in the kidney ofa subject having PKD. In certain embodiments, prior to administration, asubject is determined to have an increased level of miR-17 in thekidney. miR-17 levels may be measured from kidney biopsy material. Incertain embodiments, prior to administration, a subject is determined tohave an increased level of miR-17 in the urine or blood of the subject.

In certain embodiments, administration of a compound comprising amodified oligonucleotide complementary to miR-17 results in one or moreclinically beneficial outcomes. In certain embodiments theadministration improves kidney function in the subject. In certainembodiments the administration delays the worsening of kidney functionin the subject. In certain embodiments the administration reduces totalkidney volume in the subject. In certain embodiments the administrationslows the increase in total kidney volume in the subject. In certainembodiments, the administration reduces height-adjusted total kidneyvolume (HtTKV). In certain embodiments, the administration slows anincrease in HtTKV.

In certain embodiments the administration inhibits cyst growth in thesubject. In certain embodiments the administration slows the increase incyst growth in the subject. In some embodiments, a cyst is present inthe kidney of a subject. In some embodiments, a cyst is present in boththe liver and the kidney of the subject.

In certain embodiments the administration reduces kidney pain in thesubject. In certain embodiments the administration slows the increase inkidney pain in the subject. In certain embodiments the administrationdelays the onset of kidney pain in the subject.

In certain embodiments the administration reduces hypertension in thesubject. In certain embodiments the administration slows the worseningof hypertension in the subject. In certain embodiments theadministration delays the onset of hypertension in the subject.

In certain embodiments the administration reduces fibrosis in kidney ofthe subject. In certain embodiments the administration slows thefibrosis in the kidney of the subject.

In certain embodiments the administration delays the onset of end stagerenal disease in the subject. In certain embodiments the administrationdelays time to dialysis for the subject. In certain embodiments theadministration delays time to renal transplant for the subject. Incertain embodiments the administration improves life expectancy of thesubject.

In certain embodiments the administration reduces albuminuria in thesubject. In certain embodiments the administration slows the worseningof albuminuria in the subject. In certain embodiments the administrationdelays the onset of albuminuria in the subject. In certain embodimentsthe administration reduces hematuria in the subject. In certainembodiments the administration slows the worsening of hematuria in thesubject. In certain embodiments the administration delays the onset ofhematuria in the subject. In certain embodiments the administrationreduces blood urea nitrogen in the subject. In certain embodiments theadministration reduces creatinine in the blood of the subject. Incertain embodiments the administration improves creatinine clearance inthe subject. In certain embodiments the administration reducesalbumin:creatinine ratio in the subject. In certain embodiments theadministration improves glomerular filtration rate in the subject. Incertain embodiments, the administration slows the worsening ofglomerular filtration rate in the subject. In some embodiments, theworsening of glomerular filtration rate is assessed by calculating therate of decline of glomerular filtration rate. In certain embodimentsthe administration reduces neutrophil gelatinase-associated lipocalin(NGAL) protein in the urine of the subject. In certain embodiments theadministration reduces kidney injury molecule-1 (KIM-1) protein in theurine of the subject.

In any of the embodiments provided herein, a subject may be subjected tocertain tests to evaluate the extent of disease in the subject. Suchtests include, without limitation, measurement of total kidney volume inthe subject; measurement of hypertension in the subject; measurement ofkidney pain in the subject; measurement of fibrosis in the kidney of thesubject; measurement of blood urea nitrogen in the subject; measuringcreatinine in the blood of the subject; measuring creatinine clearancein the blood of the subject; measuring albuminuria in the subject;measuring albumin:creatinine ratio in the subject; measuring glomerularfiltration rate in the subject; measurement of neutrophilgelatinase-associated lipocalin (NGAL) protein in the urine of thesubject; and/or measurement of kidney injury molecule-1 (KIM-1) proteinin the urine of the subject

Certain Additional Therapies

Treatments for polycystic kidney disease or any of the conditions listedherein may comprise more than one therapy. As such, in certainembodiments provided herein are methods for treating a subject having orsuspected of having polycystic kidney disease comprising administeringat least one therapy in addition to administering compound comprising amodified oligonucleotide having a nucleobase sequence complementary to amiR-17.

In certain embodiments, the at least one additional therapy comprises apharmaceutical agent.

In certain embodiments, the at least one additional therapy is ananti-hypertensive agent. Anti-hypertensive agents are used to controlblood pressure of the subject.

In certain embodiments, a pharmaceutical agent is a vasopres sinreceptor 2 antagonist. In certain embodiments, a vasopressin receptor 2antagonist is tolvaptan.

In certain embodiments, pharmaceutical agents include angiotensin IIreceptor blockers (ARB). In certain embodiments, an angiotensin IIreceptor blocker is candesartan, irbesartan, olmesartan, losartan,valsartan, telmisartan, or eprosartan. In certain embodiments, an ARB isadministered at a dose ranging from 6.25 to 150 mg/m2/day. In any ofthese embodiments, an ARB is administered at a dose of 6.25 mg/m²/day,10 mg/m²/day, 12.5 mg/m²/day, 18.75 mg/m²/day, 37.5 mg/m²/day, 50mg/m²/day, or 150 mg/m²/day.

In certain embodiments, pharmaceutical agents include angiotensin IIconverting enzyme (ACE) inhibitors. In certain embodiments, an ACEinhibitor is captopril, enalapril, lisinopril, benazepril, quinapril,fosinopril, or ramipril. In certain embodiments, an ACE inhibitor isadministered at a dose ranging from 0.5 to 1 mg/m2/day, from 1 to 6mg/m2/day, from 1 to 2 mg/m2/day, from 2 to 4 mg/m2/day, or from 4 to 8mg/m2/day.

In certain embodiments, a pharmaceutical agents is a diuretic. Incertain embodiments, a pharmaceutical agent is a calcium channelblocker.

In certain embodiments, a pharmaceutical agent is a kinase inhibitor. Incertain embodiments, a kinase inhibitor is bosutinib or KD019.

In certain embodiments, a pharmaceutical agent is an adrenergic receptorantagonist.

In certain embodiments, a pharmaceutical agent is an aldosteronereceptor antagonist. In certain embodiments, an aldosterone receptorantagonist is spironolactone. In certain embodiments, spironolactone isadministered at a dose ranging from 10 to 35 mg daily. In certainembodiments, spironolactone is administered at a dose of 25 mg daily.

In certain embodiments, a pharmaceutical agent is a mammalian target ofrapamycin (mTOR) inhibitor. In certain embodiments, an mTOR inhibitor iseverolimus, rapamycin, or sirolimus.

In certain embodiments, a pharmaceutical agent is a hormone analogue. Incertain embodiments, a hormone analogue is somatostatin oradrenocorticotrophic hormone.

In certain embodiments, an additional therapy is an anti-fibrotic agent.In certain embodiments, an anti-fibrotic agent is a modifiedoligonucleotide complementary to miR-21.

In certain embodiments, an additional therapy is dialysis. In certainembodiments, an additional therapy is kidney transplant.

In certain embodiments, pharmaceutical agents include anti-inflammatoryagents. In certain embodiments, an anti-inflammatory agent is asteroidal anti-inflammatory agent. In certain embodiments, a steroidanti-inflammatory agent is a corticosteroid. In certain embodiments, acorticosteroid is prednisone. In certain embodiments, ananti-inflammatory agent is a non-steroidal anti-inflammatory drug. Incertain embodiments, a non-steroidal anti-inflammatory agent isibuprofen, a COX-I inhibitor, or a COX-2 inhibitor.

In certain embodiments, a pharmaceutical agent is a pharmaceutical agentthat blocks one or more responses to fibrogenic signals.

In certain embodiments, an additional therapy may be a pharmaceuticalagent that enhances the body's immune system, including low-dosecyclophosphamide, thymostimulin, vitamins and nutritional supplements(e.g., antioxidants, including vitamins A, C, E, beta-carotene, zinc,selenium, glutathione, coenzyme Q-10 and echinacea), and vaccines, e.g.,the immunostimulating complex (ISCOM), which comprises a vaccineformulation that combines a multimeric presentation of antigen and anadjuvant.

In certain embodiments, the additional therapy is selected to treat orameliorate a side effect of one or more pharmaceutical compositions ofthe present invention. Such side effects include, without limitation,injection site reactions, liver function test abnormalities, kidneyfunction abnormalities, liver toxicity, renal toxicity, central nervoussystem abnormalities, and myopathies. For example, increasedaminotransferase levels in serum may indicate liver toxicity or liverfunction abnormality. For example, increased bilirubin may indicateliver toxicity or liver function abnormality.

Certain MicroRNA Nucleobase Sequences

The modified oligonucleotides described herein have a nucleobasesequence that is complementary to miR-17 (SEQ ID NO: 1), or a precursorthereof (SEQ ID NO: 2). In certain embodiments, each nucleobase of themodified oligonucleotide is capable of undergoing base-pairing with anucleobase at each corresponding position in the nucleobase sequence ofmiR-17, or a precursor thereof. In certain embodiments the nucleobasesequence of a modified oligonucleotide may have one or more mismatchedbase pairs with respect to the nucleobase sequence of miR-17 orprecursor sequence, and remains capable of hybridizing to its targetsequence.

As the miR-17 sequence is contained within the miR-17 precursorsequence, a modified oligonucleotide having a nucleobase sequencecomplementary to miR-17 is also complementary to a region of the miR-17precursor.

In certain embodiments, a modified oligonucleotide consists of a numberof linked nucleosides that is equal to the length of miR-17.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is less than the length of miR-17. A modifiedoligonucleotide having a number of linked nucleosides that is less thanthe length of miR-17, wherein each nucleobase of the modifiedoligonucleotide is complementary to each nucleobase at a correspondingposition of miR-17, is considered to be a modified oligonucleotidehaving a nucleobase sequence that is fully complementary to a region ofthe miR-17 sequence. For example, a modified oligonucleotide consistingof 19 linked nucleosides, where each nucleobase is complementary to acorresponding position of miR-17 that is 22 nucleobases in length, isfully complementary to a 19 nucleobase region of miR-17. Such a modifiedoligonucleotide has 100% complementarity to (or is fully complementaryto) a 19 nucleobase segment of miR-17, and is considered to be 100%complementary to (or fully complementary to) miR-17.

In certain embodiments, a modified oligonucleotide comprises anucleobase sequence that is complementary to a seed sequence, i.e. amodified oligonucleotide comprises a seed-match sequence. In certainembodiments, a seed sequence is a hexamer seed sequence. In certain suchembodiments, a seed sequence is nucleobases 1-6 of miR-17. In certainsuch embodiments, a seed sequence is nucleobases 2-7 of miR-17. Incertain such embodiments, a seed sequence is nucleobases 3-8 of miR-17.In certain embodiments, a seed sequence is a heptamer seed sequence. Incertain such embodiments, a heptamer seed sequence is nucleobases 1-7 ofmiR-17. In certain such embodiments, a heptamer seed sequence isnucleobases 2-8 of miR-17. In certain embodiments, the seed sequence isan octamer seed sequence. In certain such embodiments, an octamer seedsequence is nucleobases 1-8 of miR-17. In certain embodiments, anoctamer seed sequence is nucleobases 2-9 of miR-17.

miR-17 is a member of a family of microRNAs known as the miR-17 family.The miR-17 family includes miR-17, miR-20a, miR-20b, miR-93, miR-106a,and miR-106b. Each member of the miR-17 family has a nucleobase sequencecomprising the nucleobase sequence 5′-AAAGUG-3,′ or the miR-17 seedregion, which is the nucleobase sequence at positions 2 through 7 of SEQID NO: 1. Additionally, each member of the miR-17 family shares somenucleobase sequence identity outside the seed region. Accordingly, amodified oligonucleotide complementary to miR-17 may target microRNAs ofthe miR-17 family, in addition to miR-17. In certain embodiments, amodified oligonucleotide targets two or more microRNAs of the miR-17family. In certain embodiments, a modified oligonucleotide targets threeor more microRNAs of the miR-17 family. In certain embodiments, amodified oligonucleotide targets four or more microRNAs of the miR-17family. In certain embodiments, a modified oligonucleotide targets fiveor more microRNAs of the miR-17 family. In certain embodiments, amodified oligonucleotide targets six of the microRNAs of the miR-17family.

In certain embodiments, a modified oligonucleotide comprises thenucleobase sequence 5′-GCACTTTG-3′ (SEQ ID NO: 3). In certainembodiments, a modified oligonucleotide comprises the nucleobasesequence 5′-AGCACTTT-3′ (SEQ ID NO: 4). In certain embodiments, amodified oligonucleotide comprises the nucleobase sequence5′-AGCACTTTG-3′(SEQ ID NO: 5). In certain embodiments, a modifiedoligonucleotide comprises the nucleobase sequence 5′-AAGCACTTTG-3′(SEQID NO: 6). In certain embodiments, a modified oligonucleotide comprisesthe nucleobase sequence 5′-TAAGCACTTTG-3′ (SEQ ID NO: 7). In certainembodiments, a modified oligonucleotide comprises the nucleobasesequence 5′-GTAAGCACTTTG-3′ (SEQ ID NO: 8). In certain embodiments, amodified oligonucleotide comprises the nucleobase sequence5′-TGTAAGCACTTTG-3′ (SEQ ID NO: 9). In certain embodiments, a modifiedoligonucleotide comprises the nucleobase sequence 5′-CTGTAAGCACTTTG-3′(SEQ ID NO: 10). In certain embodiments, a modified oligonucleotidecomprises the nucleobase sequence 5′-ACTGTAAGCACTTTG-3′ (SEQ ID NO: 11).In certain embodiments, a modified oligonucleotide comprises thenucleobase sequence 5′-CACTGTAAGCACTTTG-3′ (SEQ ID NO: 12). In certainembodiments, a modified oligonucleotide comprises the nucleobasesequence 5′-GCACTGTAAGCACTTTG-3′ (SEQ ID NO: 13). In certainembodiments, a modified oligonucleotide comprises the nucleobasesequence 5′-TGCACTGTAAGCACTTTG-3′ (SEQ ID NO: 14). In certainembodiments, a modified oligonucleotide comprises the nucleobasesequence 5′-CTGCACTGTAAGCACTTTG-3′ (SEQ ID NO: 15). In certainembodiments, a modified oligonucleotide comprises the nucleobasesequence 5′-CCTGCACTGTAAGCACTTTG-3′ (SEQ ID NO: 16). In certainembodiments, a modified oligonucleotide comprises the nucleobasesequence 5′-ACCTGCACTGTAAGCACTTTG-3′ (SEQ ID NO: 17). In certainembodiments, a modified oligonucleotide comprises the nucleobasesequence 5′-CACCTGCACTGTAAGCACTTTG-3′ (SEQ ID NO: 18). In certainembodiments, a modified oligonucleotide comprises the nucleobasesequence 5′-CTACCTGCACTGTAAGCACTTTG-3′ (SEQ ID NO: 19). In any of theseembodiments, the modified oligonucleotide consists of a nucleobasesequence selected from any one of SEQ ID Nos 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, and 18. In each nucleobase sequence, T isindependently selected from a T and a U.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence having one mismatch with respect to the nucleobase sequence ofmiR-17, or a precursor thereof. In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence having two mismatches withrespect to the nucleobase sequence of miR-17, or a precursor thereof. Incertain such embodiments, a modified oligonucleotide has a nucleobasesequence having no more than two mismatches with respect to thenucleobase sequence of miR-17, or a precursor thereof. In certain suchembodiments, the mismatched nucleobases are contiguous. In certain suchembodiments, the mismatched nucleobases are not contiguous.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is greater than the length of miR-17. In certain suchembodiments, the nucleobase of an additional nucleoside is complementaryto a nucleobase of the miR-17 stem-loop sequence. In certainembodiments, the number of linked nucleosides of a modifiedoligonucleotide is one greater than the length of miR-17. In certainsuch embodiments, the additional nucleoside is at the 5′ terminus of anoligonucleotide. In certain such embodiments, the additional nucleosideis at the 3′ terminus of an oligonucleotide. In certain embodiments, thenumber of linked nucleosides of a modified oligonucleotide is twogreater than the length of miR-17. In certain such embodiments, the twoadditional nucleosides are at the 5′ terminus of an oligonucleotide. Incertain such embodiments, the two additional nucleosides are at the 3′terminus of an oligonucleotide. In certain such embodiments, oneadditional nucleoside is located at the 5′ terminus and one additionalnucleoside is located at the 3′ terminus of an oligonucleotide. Incertain embodiments, a region of the oligonucleotide may be fullycomplementary to the nucleobase sequence of miR-17, but the entiremodified oligonucleotide is not fully complementary to miR-17. Forexample, a modified oligonucleotide consisting of 24 linked nucleosides,where the nucleobases of nucleosides 1 through 22 are each complementaryto a corresponding position of miR-17 that is 22 nucleobases in length,has a 22 nucleoside portion that is fully complementary to thenucleobase sequence of miR-17 and approximately 92% overallcomplementarity to the nucleobase sequence of miR-17.

Certain Modified Oligonucleotides

In certain embodiments, a modified oligonucleotide consists of 8 to 25linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 8 to 12 linked nucleosides. In certain embodiments, amodified oligonucleotide consists of 12 to 25 linked nucleosides. Incertain embodiments, a modified oligonucleotide consists of 15 to 25linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 15 to 19 linked nucleosides. In certain embodiments, amodified oligonucleotide consists of 15 to 16 linked nucleosides. Incertain embodiments, a modified oligonucleotide consists of 17 to 23linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 19 to 23 linked nucleosides.

In certain embodiments, a modified oligonucleotide consists of 8 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 9 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 10 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 11 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 12 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 13 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 14 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 15 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 16 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 17 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 18 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 19 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 20 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 21 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 22 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 23 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 24 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 25 linked nucleosides.

In certain embodiments, a modified oligonucleotide comprises one or more5-methylcytosines. In certain embodiments, each cytosine of a modifiedoligonucleotide comprises a 5-methylcytosine.

Certain Modifications

In certain embodiments, oligonucleotides provided herein may compriseone or more modifications to a nucleobase, sugar, and/or internucleosidelinkage, and as such is a modified oligonucleotide. A modifiednucleobase, sugar, and/or internucleoside linkage may be selected overan unmodified form because of desirable properties such as, for example,enhanced cellular uptake, enhanced affinity for other oligonucleotidesor nucleic acid targets and increased stability in the presence ofnucleases.

In certain embodiments, a modified oligonucleotide comprises one or moremodified nucleosides. In certain such embodiments, a modified nucleosideis a stabilizing nucleoside. An example of a stabilizing nucleoside is asugar-modified nucleoside.

In certain embodiments, a modified nucleoside is a sugar-modifiednucleoside. In certain such embodiments, the sugar-modified nucleosidescan further comprise a natural or modified heterocyclic base moietyand/or a natural or modified internucleoside linkage and may includefurther modifications independent from the sugar modification. Incertain embodiments, a sugar modified nucleoside is a 2′-modifiednucleoside, wherein the sugar ring is modified at the 2′ carbon fromnatural ribose or 2′-deoxy-ribose.

In certain embodiments, a 2′-modified nucleoside has a bicyclic sugarmoiety. In certain such embodiments, the bicyclic sugar moiety is a Dsugar in the alpha configuration. In certain such embodiments, thebicyclic sugar moiety is a D sugar in the beta configuration. In certainsuch embodiments, the bicyclic sugar moiety is an L sugar in the alphaconfiguration. In certain such embodiments, the bicyclic sugar moiety isan L sugar in the beta configuration.

Nucleosides comprising such bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. In certain embodiments, bicyclicnucleosides include, but are not limited to, (A) α-L-Methyleneoxy(4′-CH₂—O-2′) BNA; (B) β-D-Methyleneoxy (4′-CH₂—O-2′) BNA; (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA; (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA;(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA; (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt);(G) methylene-thio (4′-CH₂—S-2′) BNA; (H) methylene-amino(4′-CH2-N(R)-2′) BNA; (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA;(J) c-MOE (4′-CH(CH₂—OMe)-O-2′) BNA and (K) propylene carbocyclic(4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, aprotecting group, or C₁-C₁₂ alkyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, OCF₃, O—CH₃, OCH₂CH₂OCH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, andO—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, O—CH₃, and OCH₂CH₂OCH₃.

In certain embodiments, a sugar-modified nucleoside is a 4′-thiomodified nucleoside. In certain embodiments, a sugar-modified nucleosideis a 4′-thio-2′-modified nucleoside. A 4′-thio modified nucleoside has aβ-D-ribonucleoside where the 4′-O replaced with 4′-S. A4′-thio-2′-modified nucleoside is a 4′-thio modified nucleoside havingthe 2′-OH replaced with a 2′-substituent group. Suitable 2′-substituentgroups include 2′-OCH₃, 2′-O—(CH₂)₂—OCH₃, and 2′-F.

In certain embodiments, a modified oligonucleotide comprises one or moreinternucleoside modifications. In certain such embodiments, eachinternucleoside linkage of a modified oligonucleotide is a modifiedinternucleoside linkage. In certain embodiments, a modifiedinternucleoside linkage comprises a phosphorus atom.

In certain embodiments, a modified oligonucleotide comprises at leastone phosphorothioate internucleoside linkage. In certain embodiments,each internucleoside linkage of a modified oligonucleotide is aphosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide comprises one or moremodified nucleobases. In certain embodiments, a modified nucleobase isselected from 5-hydroxymethyl cytosine, 7-deazaguanine and7-deazaadenine. In certain embodiments, a modified nucleobase isselected from 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and2-pyridone. In certain embodiments, a modified nucleobase is selectedfrom 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2 aminopropyladenine, 5-propynyluraciland 5-propynylcytosine.

In certain embodiments, a modified nucleobase comprises a polycyclicheterocycle. In certain embodiments, a modified nucleobase comprises atricyclic heterocycle. In certain embodiments, a modified nucleobasecomprises a phenoxazine derivative. In certain embodiments, thephenoxazine can be further modified to form a nucleobase known in theart as a G-clamp.

In certain embodiments, a modified oligonucleotide is conjugated to oneor more moieties which enhance the activity, cellular distribution orcellular uptake of the resulting antisense oligonucleotides. In certainsuch embodiments, the moiety is a cholesterol moiety. In certainembodiments, the moiety is a lipid moiety. Additional moieties forconjugation include carbohydrates, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes. In certain embodiments, thecarbohydrate moiety is N-acetyl-D-galactosamine (GalNac). In certainembodiments, a conjugate group is attached directly to anoligonucleotide. In certain embodiments, a conjugate group is attachedto a modified oligonucleotide by a linking moiety selected from amino,hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triplebonds), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), 6-aminohexanoicacid (AHEX or AHA), substituted C1-C10 alkyl, substituted orunsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10alkynyl. In certain such embodiments, a substituent group is selectedfrom hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain such embodiments, the compound comprises a modifiedoligonucleotide having one or more stabilizing groups that are attachedto one or both termini of a modified oligonucleotide to enhanceproperties such as, for example, nuclease stability. Included instabilizing groups are cap structures. These terminal modificationsprotect a modified oligonucleotide from exonuclease degradation, and canhelp in delivery and/or localization within a cell. The cap can bepresent at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), orcan be present on both termini. Cap structures include, for example,inverted deoxy abasic caps.

Certain Pharmaceutical Compositions

Provided herein are pharmaceutical compositions. In certain embodiments,a pharmaceutical composition provided herein comprises a compoundcomprising a modified oligonucleotide consisting of 8 to 25 linkednucleosides and having a nucleobase sequence complementary to miR-17. Incertain embodiments, a pharmaceutical composition provided hereincomprises a compound consisting of a modified oligonucleotide consistingof 8 to 12 linked nucleosides and having a nucleobase sequencecomplementary to miR-17. In certain embodiments, a pharmaceuticalcomposition provided herein comprises a compound comprising a modifiedoligonucleotide consisting of 15 to 25 linked nucleosides and having anucleobase sequence complementary to miR-17. In certain embodiments, apharmaceutical composition provided herein comprises a compoundcomprising a modified oligonucleotide consisting of 17 to 23 linkednucleosides and having a nucleobase sequence complementary to miR-17.

Suitable administration routes include, but are not limited to, oral,rectal, transmucosal, intestinal, enteral, topical, suppository, throughinhalation, intrathecal, intracardiac, intraventricular,intraperitoneal, intranasal, intraocular, intratumoral, and parenteral(e.g., intravenous, intramuscular, intramedullary, and subcutaneous). Incertain embodiments, pharmaceutical intrathecals are administered toachieve local rather than systemic exposures. For example,pharmaceutical compositions may be injected directly in the area ofdesired effect (e.g., into the kidney).

In certain embodiments, a pharmaceutical composition is administered inthe form of a dosage unit (e.g., tablet, capsule, bolus, etc.). In someembodiments, a pharmaceutical compositions comprises a modifiedoligonucleotide at a dose within a range selected from 25 mg to 800 mg,25 mg to 700 mg, 25 mg to 600 mg, 25 mg to 500 mg, 25 mg to 400 mg, 25mg to 300 mg, 25 mg to 200 mg, 25 mg to 100 mg, 100 mg to 800 mg, 200 mgto 800 mg, 300 mg to 800 mg, 400 mg to 800 mg, 500 mg to 800 mg, 600 mgto 800 mg, 100 mg to 700 mg, 150 mg to 650 mg, 200 mg to 600 mg, 250 mgto 550 mg, 300 mg to 500 mg, 300 mg to 400 mg, and 400 mg to 600 mg. Incertain embodiments, such pharmaceutical compositions comprise amodified oligonucleotide in a dose selected from 25 mg, 30 mg, 35 mg, 40mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270mg, 270 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 305 mg, 310 mg, 315mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg, 350 mg, 355 mg, 360mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400 mg, 405mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, 440 mg, 445 mg, 450mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 485 mg, 490 mg, 495mg, 500 mg, 505 mg, 510 mg, 515 mg, 520 mg, 525 mg, 530 mg, 535 mg, 540mg, 545 mg, 550 mg, 555 mg, 560 mg, 565 mg, 570 mg, 575 mg, 580 mg, 585mg, 590 mg, 595 mg, 600 mg, 605 mg, 610 mg, 615 mg, 620 mg, 625 mg, 630mg, 635 mg, 640 mg, 645 mg, 650 mg, 655 mg, 660 mg, 665 mg, 670 mg, 675mg, 680 mg, 685 mg, 690 mg, 695 mg, 700 mg, 705 mg, 710 mg, 715 mg, 720mg, 725 mg, 730 mg, 735 mg, 740 mg, 745 mg, 750 mg, 755 mg, 760 mg, 765mg, 770 mg, 775 mg, 780 mg, 785 mg, 790 mg, 795 mg, and 800 mg. Incertain such embodiments, a pharmaceutical composition of the comprisesa dose of modified oligonucleotide selected from 25 mg, 50 mg, 75 mg,100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg,700 mg, and 800 mg.

In certain embodiments, a pharmaceutical agent is sterile lyophilizedmodified oligonucleotide that is reconstituted with a suitable diluent,e.g., sterile water for injection or sterile saline for injection. Thereconstituted product is administered as a subcutaneous injection or asan intravenous infusion after dilution into saline. The lyophilized drugproduct consists of a modified oligonucleotide which has been preparedin water for injection, or in saline for injection, adjusted to pH7.0-9.0 with acid or base during preparation, and then lyophilized. Thelyophilized modified oligonucleotide may be 25-800 mg of anoligonucleotide. It is understood that this encompasses 25, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mgof modified lyophilized oligonucleotide. Further, in some embodiments,the lyophilized modified oligonucleotide is an amount of anoligonucleotide within a range selected from 25 mg to 800 mg, 25 mg to700 mg, 25 mg to 600 mg, 25 mg to 500 mg, 25 mg to 400 mg, 25 mg to 300mg, 25 mg to 200 mg, 25 mg to 100 mg, 100 mg to 800 mg, 200 mg to 800mg, 300 mg to 800 mg, 400 mg to 800 mg, 500 mg to 800 mg, 600 mg to 800mg, 100 mg to 700 mg, 150 mg to 650 mg, 200 mg to 600 mg, 250 mg to 550mg, 300 mg to 500 mg, 300 mg to 400 mg, and 400 mg to 600 mg. Thelyophilized drug product may be packaged in a 2 mL Type I, clear glassvial (ammonium sulfate-treated), stoppered with a bromobutyl rubberclosure and sealed with an aluminum FLIP-OFF® overseal.

In certain embodiments, the pharmaceutical compositions provided hereinmay additionally contain other adjunct components conventionally foundin pharmaceutical compositions, at their art-established usage levels.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the oligonucleotide(s) of the formulation.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In one method, the nucleic acid is introduced into preformedliposomes or lipoplexes made of mixtures of cationic lipids and neutrallipids. In another method, DNA complexes with mono- or poly-cationiclipids are formed without the presence of a neutral lipid. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to a particular cell or tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to fat tissue. In certain embodiments, a lipidmoiety is selected to increase distribution of a pharmaceutical agent tomuscle tissue.

In certain embodiments, INTRALIPID is used to prepare a pharmaceuticalcomposition comprising an oligonucleotide. Intralipid is fat emulsionprepared for intravenous administration. It is made up of 10% soybeanoil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water forinjection. In addition, sodium hydroxide has been added to adjust the pHso that the final product pH range is 6 to 8.9.

In certain embodiments, a pharmaceutical composition provided hereincomprise a polyamine compound or a lipid moiety complexed with a nucleicacid. In certain embodiments, such preparations comprise one or morecompounds each individually having a structure defined by formula (Z) ora pharmaceutically acceptable salt thereof,

wherein each X^(a) and X^(b), for each occurrence, is independently C₁₋₆alkylene; n is 0, 1, 2, 3, 4, or 5; each R is independently H, whereinat least n+2 of the R moieties in at least about 80% of the molecules ofthe compound of formula (Z) in the preparation are not H; m is 1, 2, 3or 4; Y is O, NR², or S; R¹ is alkyl, alkenyl, or alkynyl; each of whichis optionally substituted with one or more substituents; and R² is H,alkyl, alkenyl, or alkynyl; each of which is optionally substituted eachof which is optionally substituted with one or more substituents;provided that, if n=0, then at least n+3 of the R moieties are not H.Such preparations are described in PCT publication WO/2008/042973, whichis herein incorporated by reference in its entirety for the disclosureof lipid preparations. Certain additional preparations are described inAkinc et al., Nature Biotechnology 26, 561-569 (1 May 2008), which isherein incorporated by reference in its entirety for the disclosure oflipid preparations.

In certain embodiments, pharmaceutical compositions provided hereincomprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided herein isprepared using known techniques, including, but not limited to mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or tableting processes.

In certain embodiments, a pharmaceutical composition provided herein isa liquid (e.g., a suspension, elixir and/or solution). In certain ofsuch embodiments, a liquid pharmaceutical composition is prepared usingingredients known in the art, including, but not limited to, water,glycols, oils, alcohols, flavoring agents, preservatives, and coloringagents.

In certain embodiments, a pharmaceutical composition provided herein isa solid (e.g., a powder, tablet, and/or capsule). In certain of suchembodiments, a solid pharmaceutical composition comprising one or moreoligonucleotides is prepared using ingredients known in the art,including, but not limited to, starches, sugars, diluents, granulatingagents, lubricants, binders, and disintegrating agents.

In certain embodiments, a pharmaceutical composition provided herein isformulated as a depot preparation. Certain such depot preparations aretypically longer acting than non-depot preparations. In certainembodiments, such preparations are administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. In certain embodiments, depot preparations are prepared usingsuitable polymeric or hydrophobic materials (for example an emulsion inan acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more pharmaceutical agents of the present inventionto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a sustained-release system. A non-limiting example of such asustained-release system is a semi-permeable matrix of solid hydrophobicpolymers. In certain embodiments, sustained-release systems may,depending on their chemical nature, release pharmaceutical agents over aperiod of hours, days, weeks or months.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition provided hereincomprises a modified oligonucleotide in a therapeutically effectiveamount. In certain embodiments, the therapeutically effective amount issufficient to prevent, alleviate or ameliorate symptoms of a disease orto prolong the survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, one or more modified oligonucleotides providedherein is formulated as a prodrug. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically more active form of anoligonucleotide. In certain embodiments, prodrugs are useful becausethey are easier to administer than the corresponding active form. Forexample, in certain instances, a prodrug may be more bioavailable (e.g.,through oral administration) than is the corresponding active form. Incertain instances, a prodrug may have improved solubility compared tothe corresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certain instances,such prodrugs possess superior transmittal across cell membranes, wherewater solubility is detrimental to mobility. In certain embodiments, aprodrug is an ester. In certain such embodiments, the ester ismetabolically hydrolyzed to carboxylic acid upon administration. Incertain instances the carboxylic acid containing compound is thecorresponding active form. In certain embodiments, a prodrug comprises ashort peptide (polyaminoacid) bound to an acid group. In certain of suchembodiments, the peptide is cleaved upon administration to form thecorresponding active form.

In certain embodiments, a prodrug is produced by modifying apharmaceutically active compound such that the active compound will beregenerated upon in vivo administration. The prodrug can be designed toalter the metabolic stability or the transport characteristics of adrug, to mask side effects or toxicity, to improve the flavor of a drugor to alter other characteristics or properties of a drug. By virtue ofknowledge of pharmacodynamic processes and drug metabolism in vivo,those of skill in this art, once a pharmaceutically active compound isknown, can design prodrugs of the compound (see, e.g., Nogrady (1985)Medicinal Chemistry A Biochemical Approach, Oxford University Press, NewYork, pages 388-392).

Certain Kits

The present invention also provides kits. In some embodiments, the kitscomprise one or more compounds of the invention comprising a modifiedoligonucleotide, wherein the nucleobase sequence of the oligonucleotideis complementary to the nucleobase sequence of miR-17. The compounds canhave any of the nucleoside patterns described herein. In someembodiments, the compounds can be present within a vial. A plurality ofvials, such as 10, can be present in, for example, dispensing packs. Insome embodiments, the vial is manufactured so as to be accessible with asyringe. The kit can also contain instructions for using the compounds.

In some embodiments, the kits may be used for administration of thecompound to a subject. In such instances, in addition to compoundscomprising a modified oligonucleotide complementary to miR-17, the kitcan further comprise one or more of the following: syringe, alcoholswab, cotton ball, and/or gauze pad. In some embodiments, the compoundscan be present in a pre-filled syringe (such as a single-dose syringeswith, for example, a 27 gauge, ½ inch needle with a needle guard),rather than in a vial. A plurality of pre-filled syringes, such as 10,can be present in, for example, dispensing packs. The kit can alsocontain instructions for administering the compounds comprising amodified oligonucleotide complementary to miR-17.

Certain Experimental Models

In certain embodiments, the present invention provides methods of usingand/or testing modified oligonucleotides of the present invention in anexperimental model. Those having skill in the art are able to select andmodify the protocols for such experimental models to evaluate apharmaceutical agent of the invention.

Generally, modified oligonucleotides are first tested in cultured cells.Suitable cell types include those that are related to the cell type towhich delivery of a modified oligonucleotide is desired in vivo. Forexample, suitable cell types for the study of the methods describedherein include primary or cultured cells.

In certain embodiments, the extent to which a modified oligonucleotideinterferes with the activity of miR-17 is assessed in cultured cells. Incertain embodiments, inhibition of microRNA activity may be assessed bymeasuring the levels of the microRNA. Alternatively, the level of apredicted or validated microRNA-regulated transcript may be measured. Aninhibition of microRNA activity may result in the increase in themiR-17-regulated transcript, and/or the protein encoded bymiR-17-regulated transcript. Further, in certain embodiments, certainphenotypic outcomes may be measured.

Several animal models are available to the skilled artisan for the studyof miR-17 in models of human disease. Models of polycystic kidneydisease include, but are not limited to, models with mutations and/ordeletions in Pkd1 and/or Pkd2; and models comprising mutations in othergenes. Nonlimiting exemplary models of PKD comprising mutations and/ordeletions in Pkd1 and/or Pkd2 include hypomorphic models, such as modelscomprising missense mutations in Pkd1 and models with reduced orunstable expression of Pkd2; inducible conditional knockout models; andconditional knockout models. Nonlimiting exemplary PKD models comprisingmutations in genes other than Pkd1 and Pkd2 include models withmutations in Pkhd1, Nek8, Kif3a, and/or Nphp3. PKD models are reviewed,e.g., in Shibazaki et al., Human Mol. Genet., 2008; 17(11): 1505-1516;Happe and Peters, Nat Rev Nephrol., 2014; 10(10): 587-601; and Patel etal., PNAS, 2013; 110(26): 10765-10770.

Certain Quantitation Assays

In certain embodiments, microRNA levels are quantitated in cells ortissues in vitro or in vivo. In certain embodiments, changes in microRNAlevels are measured by microarray analysis. In certain embodiments,changes in microRNA levels are measured by one of several commerciallyavailable PCR assays, such as the TaqMan□ MicroRNA Assay (AppliedBiosystems).

Modulation of microRNA activity with an anti-miR or microRNA mimic maybe assessed by microarray profiling of mRNAs. The sequences of the mRNAsthat are modulated (either increased or decreased) by the anti-miR ormicroRNA mimic are searched for microRNA seed sequences, to comparemodulation of mRNAs that are targets of the microRNA to modulation ofmRNAs that are not targets of the microRNA. In this manner, theinteraction of the anti-miR with miR-17, or miR-17 mimic with itstargets, can be evaluated. In the case of an anti-miR, mRNAs whoseexpression levels are increased are screened for the mRNA sequences thatcomprise a seed match to the microRNA to which the anti-miR iscomplementary.

Modulation of microRNA activity with an anti-miR-17 compound may beassessed by measuring the level of a mRNA target of miR-17, either bymeasuring the level of the mRNA itself, or the protein transcribedtherefrom. Antisense inhibition of a microRNA generally results in theincrease in the level of mRNA and/or protein of the mRNA target of themicroRNA.

EXAMPLES

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should in no way be construed,however, as limiting the broad scope of the invention.

Those of ordinary skill in the art will readily adopt the underlyingprinciples of this discovery to design various compounds withoutdeparting from the spirit of the current invention.

Example 1: Anti-miR-17 in a Model of Polycystic Kidney Disease

Pkhd1/cre;Pkd2^(F/F) mice spontaneously develop polycystic kidneydisease, and were used as a model of ADPKD. See Patel et al., PNAS,2013; 110(26): 10765-10770.

A modified oligonucleotide complementary to miR-17 (anti-miR-17compound) was tested in the Pkhd1/cre;Pkd2^(F/F) mouse model of ADPKD.Wild-type mice were used as control mice. An oligonucleotidecomplementary to a miRNA unrelated to miR-17 was used as a treatmentcontrol for specificity (anti-miR-control). The anti-miR-17 compound wasa fully phosphorothioated oligonucleotide 19 linked nucleosides inlength (5′-CTGCACTGTAAGCACTTTG-3′; SEQ ID NO: 15), with DNA, 2′-MOE andS-cEt sugar moieties.

From 10 to 12 days of age, sex-matched littermates of mice were treatedwith anti-miR-17 (20 mg/kg) or PBS, for a total of three daily doses. At19 days of age, the mice were treated with a fourth dose of anti-miR-17(20 mg/kg) or PBS. Anti-miR-17 was administered subcutaneously. (1)Pkhd1/cre;Pkd2^(F/F) mice, PBS administration, n=8; (2)Pkhd1/cre;Pkd2^(F/F) mice, anti-miR-control administration, n=8; (3)Pkhd1/cre;Pkd2^(F/F) mice, anti-miR-17 administration, n=8. Mice weresacrificed at 28 days, and kidney weight, cyst index, kidney function,and kidney markers measured. Statistical significance was calculated byWelch's t-test.

The mean ratio of kidney weight to body weight in Pkhd1/cre;Pkd2^(F/F)mice treated with anti-miR-17 was 17% lower than the mean ratio ofkidney weight to body weight in Pkhd1/cre;Pkd2^(F/F) mice administeredanti-miR-control or PBS only (p=0.017). Pkhd1/cre;Pkd2^(F/F) micetreated with anti-miR-17 showed a mean 6% reduction in cyst indexcompared to Pkhd1/cre;Pkd2^(F/F) mice administered anti-miR-control orPBS, although the difference was not statistically significant(p=0.072). Cyst index is a histological measurement of cystic arearelative to total kidney area. Mean serum creatinine levels inPkhd1/cre;Pkd2^(F/F) mice treated with anti-miR-17 were 25% lower thanin Pkhd1/cre;Pkd2^(F/F) mice administered anti-miR-control or PBS,although the result was not statistically significant (p=0.069). Kim1expression was reduced by 33% in Pkhd1/cre;Pkd2^(F/F) mice treated withanti-miR-17 versus anti-miR-control or PBS (p=0.024), and Nga1expression was reduced by 36% in Pkhd1/cre;Pkd2^(F/F) mice treated withanti-miR-17 versus anti-miR-control or PBS (p=0.028). Finally, bloodurea nitrogen (BUN) levels were reduced by 20% in Pkhd1/cre;Pkd2^(F/F)mice treated with anti-miR-17 versus anti-miR-control or PBS (p=0.006).BUN is a blood marker of kidney function. Higher BUN correlates withpoorer kidney function. A reduction in BUN is an indicator of reducedkidney injury and damage and improved function.

These results demonstrate that anti-miR-17 treatment leads to a positiveoutcome in Pkhd1/cre;Pkd2^(F/F) mice in the primary treatment endpoint,kidney volume. Anti-miR-17 treatment also significantly reduced BUN andexpression of kidney injury mRNA biomarkers, Kim1 and Nga1, inPkhd1/cre;Pkd2^(F/F) mice. Finally, anti-miR-17 treatment resulted in atrend toward reduced serum creatinine and reduced cyst index in thePkhd1/cre;Pkd2^(F/F) mice. These outcomes were not observed withanti-miR-control, indicating that they are specifically due to miR-17inhibition.

Example 3: Anti-miR Distribution in the Kidney of Pkhd1/Cre;Pkd2^(F/F)Mice

Oligonucleotides, including anti-miR compounds, are known to distributeto several cell types within the kidney. As reported by Chau et al., SciTransl Med., 2012, 121ra18, following administration of a Cy3-labeledanti-miR to either normal mice or mice subjected to kidney injury(unilateral ureteral obstruction, a model of interstitial fibrosis), thegreatest fluorescence intensity in the kidney was in proximal tubuleepithelium. The endothelium, pericytes, myofibroblasts, and macrophagesalso all contained detectable amounts of Cy3-labeled anti-miR. However,the glomerulus, in particular podocytes, did not appear to take upsignificant amounts of anti-miR consistent with the known distributionof chemically modified oligonucleotides (Masarjian et al.,Oligonucleotides, 2004, 14, 299-310).

To investigate the distribution of anti-miR in a mouse model ofpolycystic kidney disease, anti-miR-17 compound was administered to twodifferent groups of Pkhd1/cre;Pkd2^(F/F) mice, one starting at 10 daysof age (n=4; considered precystic) and one starting at 21 days of age(n=4; considered cystic) and to one group of wild type mice starting at21 days of age (n=4). In each group, compound was administered at 20mg/kg daily for three doses. Mice were sacrificed three days after thelast dose and kidneys were harvested and processed for histologicalanalysis. Anti-miR-17 was detected using an antibody that recognizesphosphorothioated oligonucleotides.

Sections of kidney tissue were stained with an antibody that recognizesphosphorothioated oligonucleotides as a marker for anti-miR-17 compound,or Dolichos biflorus agglutinin (DBA) as a marker for collecting ducts.In all groups, the majority of the staining was found outside thecollecting ducts, possibly in the proximal tubule epithelium.Anti-miR-17 was also delivered to collecting duct cysts even whenadministered after numerous cysts had already formed in the kidney.Staining in the collecting ducts appeared to be greater in cystscompared to normal collecting ducts, suggesting that delivery ofcompound may increase with disease state.

To confirm functional delivery, RT-qPCR was used to measure geneexpression changes induced by anti-let-7 compound. A panel of 36 let-7target genes showed significant increases in expression in bothprecystic and cystic kidneys of Pkhd1/cre;Pkd2^(F/F) mice as well asfrom wild type mice administered with anti-let-7.

These results demonstrate that anti-miR compounds can successfully bedelivered to cystic kidneys to inhibit miRNAs.

Example 3: Inhibition of miR-17 Family Members

A number of microRNAs share seed sequence identity, and are thus membersof a microRNA family. As the seed region of a microRNA is determiningfactor for target specificity, microRNA family members often regulatesimilar sets of messenger RNA targets. Outside the seed region, microRNAfamily members share varying degrees of sequence identity.

One such family is the miR-17 family, which includes miR-17, miR-20a,miR-20b, miR-93, miR-106a, and miR-106b. The individual microRNAs ofthis microRNA family are located on three different chromosomes, withinthree different microRNA clusters. miR-17 and miR-20a reside within themiR-17˜92 cluster on human chromosome 13; miR-20b and miR-106a residewithin the miR-106a˜363 cluster on the human X chromosome, and miR-93and miR-106b reside within the miR-106b˜25 cluster on human chromosome 7(FIG. 1A). Each of these three clusters contains other microRNAs thatare not members of the miR-17 family, and thus do not comprise themiR-17 2-7 seed sequence. These microRNAs, however, are members of othermiR families, as shown in FIG. 1B. The miR-17 family members are shownin FIG. 1B, with the miR-17 2-7 seed sequence in bold text. The seedsequence of the miR-18 family members (miR-18a and miR-18b) contains aone nucleobase difference relative to the miR-17 2-7 seed sequence,however outside the seed region the sequences are dissimilar.

Due to the sequence identity amongst microRNA family members, andbecause a modified oligonucleotide with less than 100% complementarityto a microRNA sequence may still inhibit the activity of that microRNA,a modified oligonucleotide with a nucleobase sequence 100% complementaryto the nucleobase sequence of a first member of the family, and which isless than 100% complementary to one or more other members of the family,may inhibit those one or more other members of the family, in additionto inhibiting the activity of the first member of the microRNA family.For example, a modified oligonucleotide with a nucleobase sequence thatis 100% complementary to miR-17 (5′-CTGCACTGTAAGCACTTTG-3′; SEQ ID NO:15), and less than 100% complementarity to other members of the miR-17family (Table 1), is expected to inhibit those other members of themiR-17 family.

TABLE 1 % Complementarity of anti-miR-17 to members of miR-17 family# of SEQ SEQUENCE (5′ TO 3′) complementary % ID microRNAmiR-17 2-7 seed in bold nucleobases Complementarity NO: miR-17CAAAGUGCUUACAGUGCAGGU 19  100% 1 AG miR-20a UAAAGUGCUUAUAGUGCAGGU 1789.5% 21 AG miR-20b CAAAGUGCUCAUAGUGCAGGU 17 89.5% 22 AG miR-93CAAAGUGCUGUUCGUGCAGGU 14 73.7% 23 AG miR-106a AAAAGUGCUUACAGUGCAGGU 1789.5% 24 AG miR-106b UAAAGUGCUGACAGUGCAGAU 16 84.2% 25

To test the inhibition of the miR-17 family members, the luciferasereporter assay was used. A luciferase reporter plasmid for each ofmiR-17, miR-20a, miR-20b, miR-93, and miR-106b was constructed, with afully complementary microRNA binding site in the 3′-UTR of theluciferase gene. For each microRNA, HeLa cells were transfected with themicroRNA mimic and its cognate luciferase reporter, followed bytransfection with anti-miR-17. Each of miR-17, miR-20a, miR-20b, miR-93,and miR-106b was inhibited by the anti-miR-17 compound, demonstratingthat the anti-miR-17 compound inhibits multiple members of the miR-17family, even when there are mismatches present between the anti-miR-17and microRNA sequences.

A separate assay, the microRNA polysome shift assay (miPSA), confirmedthat the anti-miR-17 compound directly engages three members of themiR-17 family, miR-17, miR-20b and miR-106a (Androsavich et al., NucleicAcids Research, 2015, 44: e13). The miPSA relies on the principle thatactive miRNAs bind to their mRNA targets in translationally active highmolecular weight (HMW) polysomes, whereas the inhibited miRNAs reside inthe low MW (LMW) polysomes. Treatment with anti-miR results in a shiftof the microRNA from HMW polysomes to LMW polysomes. Thus, the miPSAprovides a direct measurement of microRNA target engagement by acomplementary anti-miR. Androsavich et al. further confirmed thatnon-miR-17 family miRNAs, on the other hand, were unresponsive incomparison, with one exception: miR-18a unexpectedly showed strongcross-reactivity at higher doses (which may be explained by the miR-17and miR-18 seed sequences having only a single nucleotide A/G difference(FIG. 1).

Accordingly, treatment with an anti-miR-17 compound inhibits all membersof the miR-17 family, even where there are mismatches present betweenthe anti-miR-17 and microRNA sequences.

Example 4: miR-17 Inhibition in Human ADPKD Cysts

The effects of miR-17 inhibition were studied in primary culturesderived from human ADPKD cysts. Frozen human primary ADPKD cells wereprovided by the PKD Research Biomaterials and Cellular Models Core atthe Kansas University Medical Center (KUMC). Human primary ADPKD cellswere grown in DMEM:F12 medium (Gibco) supplemented with 5% FBS, 5 ug/mlinsulin, 5 ug/ml transferrin and 5 ng/ml sodium selenite (ITS) (Lonza),as previously described (Yamaguchi et al., Am J Physiol Renal Physiol,2010, 299: F944-951).

Proliferation Assay

At 80% confluency, human ADPKD cells were trypsinized using 1:10dilution of Trypsin in Ca⁺² and Mg⁺² free PBS. Cells were transfectedwith RNAiMAX (Life Technologies) following the manufacturer's protocolat a density of 2500 cells/well in a 96-well plate. Treatments were asfollows:

-   -   anti-miR-17 (at doses of 3 nM, 10 nM, or 30 nM; n=5 for each        treatment)    -   control oligonucleotide (at doses indicated in Table 3; n=5 for        each treatment). To vary the control treatments, two different        control groups were used. For cultures derived from donors 1-4,        3 or 5 control oligonucleotides were tested, each at a single        dose of 30 nM. For cells derived from donor 5, a single control        oligonucleotide was tested at three different doses.    -   mock-transfection with RNAiMAX (n=5)    -   PBS (n=5)

Cell viability was measured using MTT assay (Promega) on day threefollowing the manufacturer's protocol. Results are shown in Table 2. Themean for each anti-miR-17 treatment group or control oligonucleotidetreatment group is normalized to the mean for the mock treatment group.Standard error of the mean is shown. Statistical analysis was performedusing Student's t-test for pairwise comparisons or Analysis of variance(ANOVA) followed by Tukey's post hoc test for multiple comparison. Pvalues are as follows: * indicates P<0.05, **indicates P<0.01,***indicates P<0.005, and ****indicates P<0.001. For anti-miR-17treatment, P-value indicates significance relative to mock treatment.For control oligonucleotide treatment, two different P-values are shown,one indicating significance relative to mock treatment (P-value 1 inTable 2) and the other indicating significance relative to anti-miR-1730 nM treatment (P-value 2 in Table 2). N.t. indicates not tested.

Treatment with anti-miR-17 produced a dose-dependent reduction in theproliferation of cyst epithelia, relative to mock transfectiontreatment. Unlike treatment with anti-miR-17, the majority of controloligonucleotide treatments did not consistently reduce proliferation bya statistically significant amount.

As an example, treatment of cyst epithelial cultures from Donor 1 with30 nM anti-miR-17 reduced cell proliferation by 47%, relative to mocktransfection (P<0.0001), whereas treatment with control oligonucleotidesdid not reduce cell proliferation by a statistically significant amount.Further, for cyst epithelial cultures from the same donor, a comparisonof anti-miR-17 30 nM treatment to each of the three control treatmentsalso reveals a statistically significant reduction in cell proliferationby anti-miR-17 treatment (P<0.0001).

TABLE 2 Proliferation of cyst epithelia Treatment ControlOligonucleotides Mean anti-miR-17 #1 #2 #3 #4 #5 per 10 30 30 30 30 3030 Donor PBS Mock 3 nM nM nM nM nM nM nM nM Donor 1 1.94 1.00 0.98 0.890.53 1.09 0.94 1.15 n.t. n.t. mean SEM 0.03 0.05 0.03 0.02 0.01 0.040.06 0.04 p-value 1 ns ns **** ns ns ns p-value 2 **** **** **** Donor 21.00 1.00 1.00 0.94 0.78 0.97 0.91 0.83 0.64 0.97 mean SEM 0.03 0.030.03 0.02 0.03 0.02 0.03 0.05 0.04 0.01 p-value 1 ns ns *** ns ns * ****ns p-value 2 ** ns ns ns ** Donor 3 1.01 1.00 0.65 0.58 0.27 0.59 0.680.63 0.23 0.63 mean SEM 0.03 0.02 0.04 0.02 0.06 0.03 0.03 0.03 0.040.04 p-value 1 **** **** **** **** **** **** **** **** p-value 2 ******** **** ns **** Donor 4 1.36 1.00 0.75 0.62 0.17 0.74 0.67 0.57 0.400.29 mean SEM 0.05 0.07 0.03 0.05 0.01 0.04 0.03 0.02 0.02 0.02 p-value1 ** **** **** *** **** **** **** **** p-value 2 **** **** **** ** nsTreatment Control Oligonucleotide Mean anti-miR-17 #1 #1 #1 per 10 30 310 30 Donor PBS Mock 3 nM nM nM nM nM nM Donor 5 1.00 0.99 0.89 0.640.42 1.11 0.92 0.88 mean SEM 0.09 0.10 0.06 0.06 0.04 0.12 0.07 0.05p-value 1 ns ns *** ns ns ns p-value 2 **** ** **

In Vitro Cyst Formation

Human primary ADPKD cells were grown to 80% confluency and trypsinizedusing 1:10 dilution of trypsin in Ca⁺² and Mg⁺² free PBS. At day-one,cells were transfected using RNAiMAX in a six-well plate format.Treatment groups were as follows:

-   -   anti-miR-17 at doses of 1 nM, 5 nM, or 20 nM (n=3 for each dose)    -   five control oligonucleotides, each at a dose of 20 nM (n=3 for        each control oligonucleotide)    -   mock-transfection with RNAiMAX (n=3)    -   PBS        24 hours after transfection, cells were trypsinized to single        cell suspension, counted and plated in a 96-well plate at 4000        cells/well density in 130 μl of media plus Matrigel in a 4:5        ratio. Upon matrigel solidification, complete growth media was        added to the well. Media was replenished every 72 hours until 8        days post-plating when the cyst size and number was measured.

Each well was inspected for cyst proliferation using an Olympus D8 lightmicroscope. Images were recorded with an Olympus DP26 camera (OlympusCorporation) from 28 focal planes, 150 μm apart, down into the well (onthe z-axis) as twenty-eight 24-bit color TIFF images at 2448-by-1920pixels at 72 dpi. Each focal plane's image from each well was processedusing a custom R script (R Core Team 2015 R: A language and environmentfor statistical computing. R Foundation for Statistical Computing,Vienna, Austria, found at www.R-project.org.) that used the EBImageBioconductor package 15. This script detected cysts in an automated andreproducible manner and was applied to all matrigel assays in alldonors. Briefly, each image was masked for artifacts, filtered through ahigh-pass Laplacian filter, and segmented with an adaptive threshold.Detected objects in the segmented images were further processed by pixeldilation, hole-filling, and by pixel erosion. Size, radius andeccentricity statistics on each segmented object were collected. Objectswere filtered out if they had a mean radius less than or equal to 15pixels, or were of a mean object radius greater than 200 pixels, or acoefficient of variation of the radius of greater than 0.2, or if theeccentricity of the detected object was greater than 0.75. Because cystswere often larger than the distance between focal planes, counting suchcysts more than once was avoided. If a cyst object in one image fellwithin the same x- and y-coordinates of a neighboring image (e.g., onefocal plane above on the z-axis), then this was counted as the samecyst. All images in each well were processed sequentially in this manneron the z-axis. Finally, each cyst's volume was estimated by multiplyingthe mean radius of the largest object by 4/3πr³, assuming each cyst is asphere.

Results are shown in Table 3. The mean for each anti-miR-17 treatmentgroup or control oligonucleotide treatment group is normalized to themean for the mock treatment group. Standard error of the mean is shown.Statistical analysis was performed using Student's t-test for pairwisecomparisons or Analysis of variance (ANOVA) followed by Tukey's post hoctest for multiple comparison. P values are as follows: * indicatesP<0.05, **indicates P<0.01, ***indicates P<0.005, and ****indicatesP<0.001. For anti-miR-17 treatment, P-value indicates significancerelative to mock treatment. For control oligonucleotide treatment, twodifferent P-values are shown, one indicating significance relative tomock treatment (P-value 1 in Table 3) and the other indicatingsignificance relative to anti-miR-17 30 nM treatment (P-value 2 in Table3).

Treatment with anti-miR-17 produced a dose-dependent reduction in thecyst count, relative to mock transfection treatment. Unlike treatmentwith anti-miR-17, the majority of control oligonucleotide treatments didnot consistently reduce cyst count by a statistically significantamount.

As an example, treatment of cyst epithelial cultures from Donor 3 with30 nM anti-miR-17 reduced cell proliferation by 63%, relative to mocktransfection (P<0.0001), whereas treatment with control oligonucleotidesdid not consistently reduce cyst count by a statistically significantamount. Further, for cyst count from the same donor, a comparison ofanti-miR-17 30 nM treatment to each of the three control treatments alsoreveals a statistically significant reduction in cell proliferation byanti-miR-17 treatment (P<0.0001).

TABLE 3 Cyst Count Mean Treatment Cyst Control Oligonucleotides Countanti-miR-17 #1 #2 #3 #4 #5 Per 20 30 30 30 30 30 Donor PBS Mock 1 nM 5nM nM nM nM nM nM nM Donor 3 0.85 1.00 0.86 0.65 0.37 0.79 1.11 0.900.53 0.86 mean SEM 0.04 0.02 0.04 0.03 0.05 0.05 0.02 0.06 0.03 0.05p-value 1 ns *** **** * ns ns **** ns p-value 2 **** **** **** ns ****Donor 4 mean 1.22 1.00 0.69 0.46 0.24 1.08 1.02 0.88 0.44 0.75 SEM 0.040.07 0.01 0.06 0.03 0.06 0.09 0.07 0.05 0.04 p-value 1 * **** **** ns nsns **** ns p-value 2 **** **** **** ns ***

These data demonstrate that treatment with anti-miR-17 inhibits theproliferation of cysts derived from human ADPKD patients.

Example 5: Anti-miR-17 in the Pcy Model of PKD

Pcy mice bearing a mutation in Nphp3 spontaneously develop polycystickidney disease, with a slower progression of disease than that observedin the Pkhd1/cre;Pkd2^(F/F) mice. The Pcy model is used as a model ofhuman PKD, as well as a model of the nephronophthisis/medullary cystickidney disease (NPH/MCD) complex. A modified oligonucleotidecomplementary to miR-17 (anti-miR-17 compound) was tested in the Pcymouse model. Wild-type mice were used as control mice. Anoligonucleotide complementary to a miRNA unrelated to miR-17 was used asa treatment control for specificity (anti-miR-control). The anti-miR-17compound was a fully phosphorothioated oligonucleotide 19 linkednucleosides in length (5′-CTGCACTGTAAGCACTTTG-3′; SEQ ID NO: 15), withDNA, 2′-MOE and S-cEt sugar moieties.

Pcy mice (CD1-pcylusm) were obtained from PreClinOmic. From four weeksof age, Pcy mice were treated once per week with anti-miR-17 (50 mg/kgvia subcutaneous injection) or PBS, for a total of 26 doses. Theanti-miR-17 group contained 12 mice, and the PBS control group contained11 mice.

An additional control group included age-matched CD1 mice, obtained fromCharles River Laboratories. CD1 mice were subcutaneously injected withPBS one per week, for a total of 26 weeks.

At the end of the 26 week treatment period, mice were sacrificed, andone kidney was extracted and weighed and the other processed forhistological analysis. Blood urea nitrogen (BUN) and serum creatininewere measured.

For histological analysis, one kidney was perfused with cold PBS and 4%(wt/vol) paraformaldehyde and then harvested. Kidneys were fixed with 4%paraformaldehyde for 2 hours and then, embedded in paraffin forsectioning. Sagittal sections of kidneys were stained with hematoxylinand eosin (H&E). All image processing steps were automated and tookplace in freely available and open source software: An R1 script whichused functions from the EBImage Bioconductor package2 and theImageMagick3 suite of image processing tools. Kidney H&E images inAperio SVS format were converted to TIFF images, and the first frame wasretained for image analysis. First, the total kidney section area wascalculated using image segmentation. Image segmentation was similarlyused to find all internal structures including kidney cyst. A filter wasapplied to remove all objects less than a mean radius of three pixels.The cystic index is the image area associated with cysts divided by thetotal kidney areas. Cystic index was separately calculated forlongitudinal and transverse kidney sections for each individual animal.Combined cystic index of individual animals were compared for eachtreatment groups.

Results from PBS-treated CD1 mice were used to provide a benchmark foreach parameter (kidney weight/body weight ratio, cystic index, bloodurea nitrogen and serum creatinine) in a non-disease model. As expected,treated CD1 mice did not exhibit any pathologies associated with PKD.

The mean ratio of kidney weight to body weight in the Pcy mice treatedwith anti-miR-17 was 19% lower than the mean ratio of kidney weight tobody weight in the Pcy mice administered PBS only (p=0.0003) (FIG. 2A).Pcy mice treated with anti-miR-17 showed a mean 28% reduction in cystindex compared to Pcy mice administered PBS only (p=0.008) (FIG. 2B). Nosignificant changes in BUN or serum creatinine were observed. P-valuesof 1-way ANOVA analysis following Dunnett's multiple comparisoncorrections are shown.

These data demonstrate, in an additional model of PKD, that treatmentwith anti-miR-17 leads to a reduction in kidney weight and cyst index.

1. A method of treating polycystic kidney disease comprisingadministering to a subject in need thereof a compound comprising amodified oligonucleotide consisting of 8 to 25 linked nucleosides,wherein the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-17.
 2. The method of claim 1, wherein the subjecthas polycystic kidney disease.
 3. The method of claim 1, wherein thesubject is suspected of having polycystic kidney disease.
 4. The methodof claim 1 wherein the subject has been diagnosed as having polycystickidney disease prior to administering the modified oligonucleotide. 5.The method of claim 1 wherein the subject, prior to administration ofthe modified oligonucleotide, was determined to have an increased levelof miR-17 in the kidney, urine or blood of the subject.
 6. The method ofclaim 1, wherein the polycystic kidney disease is autosomal recessivepolycystic kidney disease or autosomal dominant polycystic kidneydisease.
 7. The method of claim 1, wherein the polycystic kidney diseaseis autosomal dominant polycystic kidney disease.
 8. The method of claim1, wherein the subject has a mutation selected from a mutation in thePKD1 gene or a mutation in the PKD2 gene.
 9. The method of claim 1,wherein the subject has increased total kidney volume.
 10. The method ofclaim 1, wherein the subject has hypertension.
 11. The method of claim1, wherein the subject has impaired kidney function.
 12. The method ofclaim 1, wherein the subject is in need of improved kidney function. 13.The method of claim 1, wherein the administering: a) improves kidneyfunction in the subject; b) delays the worsening of kidney function inthe subject; c) reduces total kidney volume in the subject; d) slows theincrease in total kidney volume in the subject; e) inhibits cyst growthin the subject; f) slows the increase in cyst growth in the subject; g)reduces kidney pain in the subject; h) slows the increase in kidney painin the subject; i) delays the onset of kidney pain in the subject; j)reduces hypertension in the subject; k) slows the worsening ofhypertension in the subject; l) delays the onset of hypertension in thesubject; m) reduces fibrosis in the kidney of the subject; n) slows theworsening of fibrosis in the kidney of the subject; o) delays the onsetof end stage renal disease in the subject; p) delays time to dialysisfor the subject; q) delays time to renal transplant for the subject;and/or r) improves life expectancy of the subject.
 14. The method ofclaim 1, wherein the administering: s) reduces albuminuria in thesubject; t) slows the worsening of albuminuria in the subject; u) delaysthe onset of albuminuria in the subject; v) reduces hematuria in thesubject; w) slows the worsening of hematuria in the subject; x) delaysthe onset of hematuria in the subject; y) reduces blood urea nitrogen inthe subject; z) reduces creatinine in the blood of the subject; aa)improves creatinine clearance in the subject; bb) reducesalbumin:creatinine ratio in the subject; cc) improves glomerularfiltration rate in the subject; dd) slows the worsening of glomerularfiltration rate in the subject; ee) reduces neutrophilgelatinase-associated lipocalin (NGAL) protein in the urine of thesubject; and/or ff) reduces kidney injury molecule-1 (KIM-1) protein inthe urine of the subject.
 15. The method of claim 1, comprising: gg)measuring total kidney volume in the subject; hh) measuring hypertensionin the subject; ii) measuring kidney pain in the subject; jj) measuringfibrosis in the kidney of the subject; kk) measuring blood urea nitrogenin the blood of the subject; ll) measuring creatinine in the blood ofthe subject; mm) measuring creatinine clearance in the subject; nn)measuring albuminuria in the subject; oo) measuring albumin:creatinineratio in the subject; pp) measuring glomerular filtration rate in thesubject; qq) measuring neutrophil gelatinase-associated lipocalin (NGAL)protein in the urine of the subject; and/or rr) measuring kidney injurymolecule-1 (KIM-1) protein in the urine of the subject.
 16. The methodof claim 9, wherein the total kidney volume is height-adjusted kidneyvolume.
 17. The method of claim 13, wherein the cyst is present in oneor more kidneys in the subject.
 18. The method of claim 13, wherein thecyst is present in the liver of the subject.
 19. The method of claim 1,comprising administering at least one additional therapy that is ananti-hypertensive agent.
 20. The method of claim 1, comprisingadministering at least one additional therapy selected from anangiotensin II converting enzyme (ACE) inhibitor, an angiotensin IIreceptor blocker (ARB), a diuretic, a calcium channel blocker, a kinaseinhibitor, an adrenergic receptor antagonist, a vasodilator, abenzodiazepine, a renin inhibitor, an aldosterone receptor antagonist,an endothelin receptor blocker, an mammalian target of rapamycin (mTOR)inhibitor, a hormone analogue, a vasopressin receptor 2 antagonist, analdosterone receptor antagonist, dialysis, and kidney transplant. 21.The method of claim 20 wherein the angiotensin II converting enzyme(ACE) inhibitor is selected from captopril, enalapril, lisinopril,benazepril, quinapril, fosinopril, and ramipril.
 22. The method of claim20 wherein the angiotensin II receptor blocker (ARB) is selected fromcandesartan, irbesartan, olmesartan, losartan, valsartan, telmisartan,and eprosartan.
 23. The method of claim 20 wherein the vasopressinreceptor 2 antagonist is tolvaptan.
 24. The method of claim 20, whereinthe aldosterone receptor antagonist is spironolactone.
 25. The method ofclaim 20, wherein the kinase inhibitor is selected from bosutinib andKD019.
 26. The method of claim 20, wherein the mTOR inhibitor isselected from everolimus, rapamycin, and sirolimus.
 27. The method ofclaim 20, the hormone analogue is selected from somatostatin andadrenocorticotrophic hormone.
 28. The method of claim 1, wherein thenucleobase sequence of the modified oligonucleotide is at least 90%complementary, is at least 95% complementary, or is 100% complementaryto the nucleobase sequence of miR-17 (SEQ ID NO: 1).
 29. The method ofclaim 1, wherein the nucleobase sequence of the modified oligonucleotidecomprises the nucleobase sequence 5′-GCACTTTG-3′ (SEQ ID NO: 3), whereineach T in the nucleobase sequence is independently selected from a T anda U.
 30. The method of claim 1, wherein the modified oligonucleotideconsists of 8 to 12 linked nucleosides.
 31. The method of claim 1,wherein the modified oligonucleotide consists of 12 to 25 linkednucleosides.
 32. The method of claim 1, wherein the modifiedoligonucleotide consists of 15 to 25 linked nucleosides.
 33. The methodof claim 1, wherein the modified oligonucleotide consists of 17 to 23linked nucleosides.
 34. The method of claim 1, wherein the modifiedoligonucleotide consists of 8, 9, 10, 11 or 12 linked nucleosides. 35.The method of claim 1, wherein the modified oligonucleotide consists of12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 linkednucleosides.
 36. The method of claim 1, wherein the modifiedoligonucleotide consists of 15, 16, 17, 18, 19, 20, 21, or 22 linkednucleosides.
 37. The method of claim 1, wherein the modifiedoligonucleotide consists of 17, 18, 19, 20, 21, 22, or 23 linkednucleosides.
 38. The method of claim 1, wherein the modifiedoligonucleotide comprises at least one modified nucleoside.
 39. Themethod of claim 38, wherein the modified nucleoside is selected from anS-cEt nucleoside, a 2′-O-methoxyethyl nucleoside, and an LNA nucleoside.40. The method of claim 1, wherein the modified oligonucleotidecomprises at least one modified internucleoside linkage.
 41. The methodof claim 1, wherein each internucleoside linkage of the modifiedoligonucleotide is a modified internucleoside linkage.
 42. The method ofclaim 40, wherein the modified internucleoside linkage is aphosphorothioate internucleoside linkage.
 43. The method of claim 1,wherein the compound consists of the modified oligonucleotide.
 44. Themethod of claim 1, comprising administering a therapeutically effectiveamount of the compound.
 45. (canceled)